Publications by authors named "Behrouz Vaziri"

39 Publications

Rabies virus matrix protein targets host actin cytoskeleton: a protein-protein interaction analysis.

Pathog Dis 2021 Jan;79(1)

Biotechnology Research Center, Pasteur Institute of Iran, Tehran, 1316943551, Iran.

Multifunctional matrix protein (M) of rabies virus (RABV) plays essential roles in the pathogenesis of rabies infection. Identification of M protein interacting partners in target hosts could help to elucidate the biological pathways and molecular mechanisms involved in the pathogenesis of this virus. In this study, two-dimensional Far-western blotting (2D-Far-WB) technique was applied to find possible matrix protein partners in the rat brainstem. Recombinant RABV M was expressed in Pichia pastoris and was partially purified. Subsequently, 2D-Far-WB-determined six rat brainstem proteins interacted with recombinant M proteins that were identified by mass spectrometry. Functional annotation by gene ontology analysis determined these proteins were involved in the regulation of synaptic transmission processes, metabolic process and cell morphogenesis-cytoskeleton organization. The interaction of viral M protein with selected host proteins in mouse Neuro-2a cells infected with RABV was verified by super-resolution confocal microscopy. Molecular docking simulations also demonstrated the formation of RABV M complexes. However, further confirmation with co-immunoprecipitation was only successful for M-actin cytoplasmic 1 interaction. Our study revealed actin cytoplasmic 1 as a binding partner of M protein, which might have important role(s) in rabies pathogenesis.
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http://dx.doi.org/10.1093/femspd/ftaa075DOI Listing
January 2021

Systematically gap-filling the genome-scale metabolic model of CHO cells.

Biotechnol Lett 2021 Jan 10;43(1):73-87. Epub 2020 Oct 10.

Department of Bioengineering, University of California, San Diego, USA.

Objective: Chinese hamster ovary (CHO) cells are the leading cell factories for producing recombinant proteins in the biopharmaceutical industry. In this regard, constraint-based metabolic models are useful platforms to perform computational analysis of cell metabolism. These models need to be regularly updated in order to include the latest biochemical data of the cells, and to increase their predictive power. Here, we provide an update to iCHO1766, the metabolic model of CHO cells.

Results: We expanded the existing model of Chinese hamster metabolism with the help of four gap-filling approaches, leading to the addition of 773 new reactions and 335 new genes. We incorporated these into an updated genome-scale metabolic network model of CHO cells, named iCHO2101. In this updated model, the number of reactions and pathways capable of carrying flux is substantially increased.

Conclusions: The present CHO model is an important step towards more complete metabolic models of CHO cells.
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http://dx.doi.org/10.1007/s10529-020-03021-wDOI Listing
January 2021

Physicochemical Characterization of Altebrel™, a Proposed Etanercept Biosimilar.

Iran J Biotechnol 2019 Dec 1;17(4):e2470. Epub 2019 Dec 1.

Department of Biotechnology, Pasteur Institute of Iran, Tehran, Iran.

Background: Etanercept is prescribed for the rapid and effective treatment of chronic immune-mediated inflammatory disorders. Due to the expiration of etanercept patents and worldwide demand for comparable and more affordable substitutes, several biosimilars of etanercept have been approved in different countries and new ones are in the process of approval.

Objectives: In the present study, Altebrel™ as an etanercept proposed biosimilar was investigated in a side by side comparison using various orthogonal analytical methods.

Materials And Methods: Three batches of the Altebrel™ and Enbrel samples were used for the study. Several physicochemical properties of samples were compared according to international guidelines, incliding; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Capillary electrophoresis sodium dodecyl sulfate (CE-SDS), size exclusion high performance liquid chromatography (SE-HPLC), hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC) and its biological activity was evaluated using surface plasmon resonance affinity analysis and tumor necrosis factor alpha (TNFα) neutralization biological assay. Amino acid analysis was applied to check the primary sequence and far-UV circular dichroism (CD) spectroscopy investigated the secondary structure.

Results: The obtained results indicated a high degree of similarity between Altebrel™ and Enbrel. Results of SDS-PAGE, CE-SDS, HIC-HPLC and SE-HPLC implied a comparable pattern of size variants for all samples. Based on the data achieved via bioactivity assays and SPR analysis, the functional property of Altebrel™ was proved comparable to that of the reference product. Moreover, amino acid analysis indicated similar primary structure and circular dichroism study implied a similar secondary structure for Altebrel™ and Enbrel.

Conclusion: Overall, our data provide analytical evidence for structural and functional similarity between Altebrel™ and Enbrel.
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http://dx.doi.org/10.30498/IJB.2019.99581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357702PMC
December 2019

Proteomics Analysis of Trastuzumab Toxicity in the H9c2 Cardiomyoblast Cell Line and its Inhibition by Carvedilol.

Curr Pharm Biotechnol 2020 ;21(13):1377-1385

Protein Chemistry and Proteomics Laboratory, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

Objective: Heart dysfunctions are the major complications of trastuzumab in patients with Human Epidermal growth factor Receptor-2 (HER2)-positive breast cancers.

Methods: In this study, the cytotoxicity of trastuzumab on H9c2 cardiomyoblasts was demonstrated, and the proteome changes of cells were investigated by a tandem mass tagging quantitative approach. The Differentially Abundant Proteins (DAPs) were identified and functionally enriched.

Results: We determined that carvedilol, a non-selective beta-blocker, could effectively inhibit trastuzumab toxicity when administrated in a proper dose and at the same time. The proteomics analysis of carvedilol co-treated cardiomyoblasts showed complete or partial reversion in expressional levels of trastuzumab-induced DAPs.

Conclusion: Downregulation of proteins involved in the translation biological process is one of the most important changes induced by trastuzumab and reversed by carvedilol. These findings provide novel insights to develop new strategies for the cardiotoxicity of trastuzumab.
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http://dx.doi.org/10.2174/1389201021666200515135548DOI Listing
December 2020

A metabolic network-based approach for developing feeding strategies for CHO cells to increase monoclonal antibody production.

Bioprocess Biosyst Eng 2020 Aug 24;43(8):1381-1389. Epub 2020 Mar 24.

Protein Chemistry and Proteomics Laboratory, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

Chinese hamster ovary (CHO) cells are the main workhorse in the biopharmaceutical industry for the production of recombinant proteins, such as monoclonal antibodies. To date, a variety of metabolic engineering approaches have been used to improve the productivity of CHO cells. While genetic manipulations are potentially laborious in mammalian cells, rational design of CHO cell culture medium or efficient fed-batch strategies are more popular approaches for bioprocess optimization. In this study, a genome-scale metabolic network model of CHO cells was used to design feeding strategies for CHO cells to improve monoclonal antibody (mAb) production. A number of metabolites, including threonine and arachidonate, were suggested by the model to be added into cell culture medium. The designed composition has been experimentally validated, and then optimized, using design of experiment methods. About a two-fold increase in the total mAb expression has been observed using this strategy. Our approach can be used in similar bioprocess optimization problems, to suggest new ways of increasing production in different cell factories.
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http://dx.doi.org/10.1007/s00449-020-02332-6DOI Listing
August 2020

In Vivo Evaluation of Carvedilol Cardiac Protection Against Trastuzumab Cardiotoxicity.

Drug Res (Stuttg) 2020 Apr 19;70(4):165-169. Epub 2020 Feb 19.

Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

Cardiac dysfunction is a major side effect of trastuzumab therapy for patients with HER2-positive breast cancer. Beta blockers, such as carvedilol, have been used for protection of trastuzumab cardiotoxicity but there is no definitive conclusive clinical report on their efficacy. In the present study, the preservability effects of carvedilol on trastuzumab-induced left ventricular (LV) dysfunction and the reversibility of trastuzumab-induced cardiotoxicity were evaluated in Wistar rats by echocardiography method. We showed that trastuzumab treatment of rats could induce the LV dysfunction through increasing the LV internal systolic diameter (LVIDs), increasing the end-systolic volume (ESV), decreasing the ejection fraction (EF), and decreasing the fractional shortening (FS). These parameters were not reversed after 14 days of stopping trastuzumab administration. Interestingly, carvedilol improved LVIDs, ESV, EF, and FS. Collectively, the results of this study have verified clinical observations which simultaneously administration of carvedilol may be considered as a possible therapeutic strategy to prevent trastuzumab-mediated LV dysfunction.
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http://dx.doi.org/10.1055/a-1110-7034DOI Listing
April 2020

Structural and In Vitro Functional Comparability Analysis of Altebrel™, a Proposed Etanercept Biosimilar: Focus on Primary Sequence and Glycosylation.

Pharmaceuticals (Basel) 2019 Jan 17;12(1). Epub 2019 Jan 17.

Mass Spectrometric Proteomics, Institute of Clinical Chemistry and Laboratory Medicine, Campus Forschung, N27 Raum 00.008, Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany.

The demand for reliable comparability studies of biosimilars grows with their increased market share. These studies focus on physicochemical, structural, functional and clinical properties to ensure that a biosimilar has no significant differences to the originator product and can be released into the market without extensive clinical trials. In the current study, Enbrel (etanercept, the originator) and Altebrel™ (the proposed biosimilar) underwent direct comparison. "Bottom-up" mass spectrometric analysis was used for primary sequence analysis, evaluation of N/O-glycosylation sites and quantification of methionine oxidation. N/O-glycans were analyzed after permethylation derivatization and the effect of N-glycans on in-vitro functionality of etanercept was assayed. Three enzyme peptide mapping resulted in complete identification of the primary structure. It was confirmed that total ion chromatograms are valuable datasets for the analysis of the primary structure of biodrugs. New N/O-glycan structures were identified and all the N-glycans were quantified. Finally, investigation of the functional properties of N-deglycosylated and non-modified etanercept samples using surface plasmon resonance analysis and in-vitro bioassay showed that N-glycosylation has no significant effect on its in-vitro functionality. Analysis of etanercept and its biosimilar, revealed a high similarity in terms of glycosylation, primary structure and in-vitro functionality.
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http://dx.doi.org/10.3390/ph12010014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6469174PMC
January 2019

Quantitative Proteomic Analysis of Cellular Responses to a Designed Amino Acid Feed in a Monoclonal Antibody Producing Chinese Hamster Ovary Cell Line

Iran Biomed J 2018 11 21;22(6):385-93. Epub 2018 Apr 21.

Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

Background: Chinese hamster ovary (CHO) cell line is considered as the most common cell line in the biopharmaceutical industry because of its capability in performing efficient post-translational modifications and producing the recombinant proteins, which are similar to natural human proteins. The optimization of the upstream process via different feed strategies has a great impact on the target molecule expression and yield.

Methods: To determine and understand the molecular events beneath the feed effects on the CHO cell, a label-free quantitative proteomic analysis was applied. The proteome changes followed by the addition of a designed amino acid feed to the monoclonal antibody producing CHO cell line culture medium were investigated.

Results: The glutathione synthesis, the negative regulation of the programmed cell death, proteasomal catabolic process, and the endosomal transport pathway were up-regulated in the group fed with a designed amino acid feed compared to the control group.

Conclusion: Our findings could be helpful to identify new targets for metabolic engineering.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6305810PMC
November 2018

A systems medicine approach for finding target proteins affecting treatment outcomes in patients with non-Hodgkin lymphoma.

PLoS One 2017 11;12(9):e0183969. Epub 2017 Sep 11.

Human Antibody Lab, Innovation Center, Pasteur Institute of Iran, Tehran, Iran.

Autoantibody profiling with a systems medicine approach can help identify critical dysregulated signaling pathways (SPs) in cancers. In this way, immunoglobulins G (IgG) purified from the serum samples of 92 healthy controls, 10 pre-treated (PR) non-Hodgkin lymphoma (NHL) patients, and 20 NHL patients who underwent chemotherapy (PS) were screened with a phage-displayed random peptide library. Protein-protein interaction networks of the PR and PS groups were analyzed and visualized by Gephi. The results indicated AXIN2, SENP2, TOP2A, FZD6, NLK, HDAC2, HDAC1, and EHMT2, in addition to CAMK2A, PLCG1, PLCG2, GRM5, GRIN2B, GRIN2D, CACNA2D3, and SPTAN1 as hubs in 11 and 7 modules of PR and PS networks, respectively. PR- and PS-specific hubs were evaluated in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome databases. The PR-specific hubs were involved in Wnt SP, signaling by Notch1 in cancer, telomere maintenance, and transcriptional misregulation. In contrast, glutamate receptor SP, Fc receptor-related pathways, growth factors-related SPs, and Wnt SP were statistically significant enriched pathways, based on the pathway analysis of PS hubs. The results revealed that the most PR-specific proteins were associated with events involved in tumor development, while chemotherapy in the PS group was associated with side effects of drugs and/or cancer recurrence. As the findings demonstrated, PR- and PS-specific proteins in this study can be promising therapeutic targets in future studies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0183969PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593188PMC
October 2017

Development of a novel nano-sized anti-VEGFA nanobody with enhanced physicochemical and pharmacokinetic properties.

Artif Cells Nanomed Biotechnol 2018 Nov 25;46(7):1402-1414. Epub 2017 Aug 25.

f Department of Civil Engineering , Sharif University of Technology , Tehran , Iran.

Since physiological and pathological processes occur at nano-environments, nanotechnology has considered as an efficient tool for designing of next generation specific biomolecules with enhanced pharmacodynamic and pharmacodynamic properties. In the current investigation, by control of the size and hydrodynamic volume at the nanoscale, for the first time, physicochemical and pharmacokinetic properties of an anti-VEGFA nanobody was remarkably improved by attachment of a Proline-Alanine-Serine (PAS) rich sequence. The results elucidated unexpected impressive effects of PAS sequence on physicochemical properties especially on size, hydrodynamics radius, and even solubility of nanobody. CD analysis revealed an increment in random coil structure of the PASylated protein in comparison to native one without any change in charge state or binding kinetic parameters of nanobody assessed by isoelectric focusing and surface plasmon resonance measurements, respectively. In vitro biological activities of nanobody were not affected by coupling of the PAS sequence. In contrast, the terminal half-life was significantly increased by a factor of 14 for the nanobody-PAS after single dose IV injection to the mice. Our study demonstrated that the control of size in the design of small therapeutic proteins has a promising effect on the stability and solubility, in addition to their physiochemical and pharmacokinetic properties. The designed new anti-VEGFA nanobody could promise a better therapeutic agent with a long administration intervals and lower dose, which in turn leads to a better patient compliance. Size adjustment of an anti-VEGF nanobody at the nanoscale by the attachment of a natural PAS polymer remarkably improves physicochemical properties, as well as a pharmacokinetic profile without any change in biological activity of the miniaturized antibody.
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http://dx.doi.org/10.1080/21691401.2017.1369426DOI Listing
November 2018

Variable spontaneous mutation rate in clinical strains of multidrug-resistant Acinetobacter baumannii and differentially expressed proteins in a hypermutator strain.

Mutat Res 2017 08 8;800-802:37-45. Epub 2017 Jun 8.

Department of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.

Background: The emergence of multidrug resistant Acinetobacter baumannii within hospitals poses a significant threat to patients. The inherent rate of mutation of these strains has not been described nor has the mechanism by which drug resistance arises.

Methods: Here, we determined the spontaneous mutation rates in 93 clinical strains of A. baumannii using fluctuation analysis. To rule out the clonal relatedness of hypermutator strains, pulsed-field gel electrophoresis (PFGE) was conducted. Using a combination of two-dimensional gel electrophoresis (2-DE) and MALDI-TOF mass spectrometry, the differentially expressed proteins of a hypermutator and a reference strain were identified.

Results: The spontaneous mutation rate of multi-drug resistant A. baumannii strains varied broadly from 0 to 2.1×10 mutation per cell division. The mutation rate in three multidrug resistant A. baumannii (MDR-AB) strains was found to be 1.63×10 (95% confidence interval (CI): 1×10-2×10), 2.1×10 (95% CI: 2×10 - 3×10), and 1.78×10 (95% CI: 9.29×10 2.95×10), consistent with a hypermutator phenotype. This rate is approximately 1000-fold higher than the average mutation rate in other MDR-ABs. PFGE of the three hypermutator strains indicate that they belong to distinct clones. Proteomic analysis of one hypermutator strain revealed 31 differentially expressed proteins including three with sizes of 51.2, 20.9, and 11.9kDa, which corresponded to a serine protease, a polyisoprenoid-binding protein, and the peptidoglycan binding protein, LysM. The serine protease was expressed only in the hypermutator strain, whereas the polyisoprenoid-binding protein and the peptidoglycan binding protein LysM were down-regulated 1.6 and 3-fold, respectively, in the hypermutators strain.

Conclusion: Hypermutator A. baumannii strains occur with a low, but appreciable frequency among clinical multi-drug resistant isolates. The presence of hypermutator clinical isolates raises concerns that they may contribute to the failure of antibiotic treatment in infected patients and confound the interpretation of in vitro antibiotic susceptibility testing. The differentially expressed proteins involved in biofilm suppression and oxidative stress response, may represent adaptations derived from the hypermutator phenotype, a hypothesis that needs further testing.
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http://dx.doi.org/10.1016/j.mrfmmm.2017.06.002DOI Listing
August 2017

Evaluating the expression profile and stability of different UCOE containing vector combinations in mAb-producing CHO cells.

BMC Biotechnol 2017 02 22;17(1):18. Epub 2017 Feb 22.

Biotechnology Research Center, Pasteur Institute of Iran, Tehran, 1316943551, Iran.

Background: As the demand for monoclonal antibodies (mAb) increases, more efficient expression methods are required for their manufacturing process. Transcriptional gene silencing is a common phenomenon in recombinant cell lines which leads to expression reduction and instability. There are reports on improved antibody expression in ubiquitous chromatin opening element (UCOE) containing both heavy and light chain gene constructs. Here we investigate the impact of having these elements as part of the light chain, heavy chain or both genes during cell line development. In this regard, non-UCOE and UCOE vectors were constructed and stable Chinese hamster ovary (CHO) cell pools were generated by different vector combinations.

Results: Expression analysis revealed that all UCOE cell pools had higher antibody yields compared to non-UCOE cells, Moreover the most optimal expression was obtained by cells containing just the UCOE on heavy chain. In terms of stability, it was shown that the high level of expression was kept consistence for more than four months in these cells whereas the expression titers were reduced in the other UCOE pools.

Conclusions: In conclusion, UCOE significantly enhanced the level and stability of antibody expression and the use of this element with heavy chain provided more stable cell lines with higher production level.
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http://dx.doi.org/10.1186/s12896-017-0330-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5322649PMC
February 2017

Scale up and pharmacokinetic study of a novel mutated chimeric tissue plasminogen activator (mt-PA) in rats.

Sci Rep 2017 02 22;7:43028. Epub 2017 Feb 22.

Biotechnology Research Center, Pasteur Institute of Iran, Pasteur Avenue, Tehran, Iran.

Because of high mortality caused by cardiovascular diseases, various fibrinolytic agents with diverse pharmacokinetic and pharmacodynamic properties have been developed. A novel mutated chimeric tissue plasminogen activator (mt-PA) was developed by the removal of first three domains of t-PA, insertion of GHRP sequence and mutation towards resistance to plasminogen activator inhibitor-1 (PAI-1). Mt-PA protein was expressed in Expi293F cells. The expression level of mt-PA was found to be 5000 IU/mL. Following purification, the pharmacokinetic properties of mt-PA were evaluated in three doses in rats. Data related to mt-PA were best fitted to two compartment model. With the increase in dose, the Area Under the plasma concentration-time Curve (AUC) increased. The elimination half-life (t) of mt-PA was in the range of 19.1-26.1 min in three doses while that of Alteplase was 8.3 min. The plasma clearance (CLp) of mt-PA ranged from 3.8 to 5.9 mL/min in three doses, which was several times lower than that of Alteplase (142.6 mL/min). The mean residence time (MRT) of mt-PA ranged from 23.3-31.8 min in three doses, which was 4-5 times greater than that of Alteplase (6 min). Mt-PA showed extended half-life and mean residence time and is a good candidate for further clinical studies.
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http://dx.doi.org/10.1038/srep43028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5320447PMC
February 2017

Proteomics Profiling of Chimeric-Truncated Tissue Plasminogen activator Producing- Chinese Hamster Ovary Cells Cultivated in a Chemically Defined Medium Supplemented with Protein Hydrolysates

Iran Biomed J 2017 05 10;21(3):154-66. Epub 2017 Apr 10.

Protein Chemistry Unit, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

Background: Culture media enrichment through the addition of protein hydrolysates is beneficial for achieving higher protein expression.

Methods: In this study, designing the optimum mixture of four soy and casein-derived hydrolysates was successfully performed by design of experiment and specific productivity increased in all predicted combinations. Protein profile of recombinant CHO (rCHO) cells producing tissue plasminogen activator in a serum-free medium (SFM) supplemented with designed hydrolysate additives was compared to that of rCHO cells cultivated in SFM.

Results: Identification of differentially expressed proteins using two-dimensional gel electrophoresis coupled with MALDI-TOF/TOF revealed the role of energy metabolism related proteins and importance of prevention of oxidative stress by this special media enrichment strategy. Up-regulation of mitochondrial enzymes, pyruvate dehydrogenase E1 and Peroxiredoxin-III, as well as other proteins involved in metabolic pathways, and uridine monophosphate/cytidine monophosphate kinase indicated higher metabolic activity. Furthermore, along with antioxidant effect of peptones, proteins with antioxidant function such as ferritin and peroxiredoxin-III were up-regulated.

Conclusion: Understanding molecular mechanisms involved in enhancement of protein expression can provide new approaches for efficiently engineering rCHO cell. These results support the competence of proteomics studies in finding new insights to biochemical pathways for a knowledge-based optimization of media compositions.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5392218PMC
http://dx.doi.org/10.18869/acadpub.ibj.21.3.154DOI Listing
May 2017

Main Quality Attributes of Monoclonal Antibodies and Effect of Cell Culture Components

Iran Biomed J 2017 05 20;21(3):131-41. Epub 2017 Apr 20.

Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

The culture media optimization is an inevitable part of upstream process development in therapeutic monoclonal antibodies (mAbs) production. The quality by design (QbD) approach defines the assured quality of the final product through the development stage. An important step in QbD is determination of the main quality attributes. During the media optimization, some of the main quality attributes such as glycosylation pattern, charge variants, aggregates, and low-molecular-weight species, could be significantly altered. Here, we provide an overview of how cell culture medium components affects the main quality attributes of the mAbs. Knowing the relationship between the culture media components and the main quality attributes could be successfully utilized for a rational optimization of mammalian cell culture media for industrial mAbs production.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5392216PMC
http://dx.doi.org/10.18869/acadpub.ibj.21.3.131DOI Listing
May 2017

Systems Biomedicine of Rabies Delineates the Affected Signaling Pathways.

Front Microbiol 2016 7;7:1688. Epub 2016 Nov 7.

Drug Design and Bioinformatics Unit, Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran Tehran, Iran.

The prototypical neurotropic virus, rabies, is a member of the Rhabdoviridae family that causes lethal encephalomyelitis. Although there have been a plethora of studies investigating the etiological mechanism of the rabies virus and many precautionary methods have been implemented to avert the disease outbreak over the last century, the disease has surprisingly no definite remedy at its late stages. The psychological symptoms and the underlying etiology, as well as the rare survival rate from rabies encephalitis, has still remained a mystery. We, therefore, undertook a systems biomedicine approach to identify the network of gene products implicated in rabies. This was done by meta-analyzing whole-transcriptome microarray datasets of the CNS infected by strain CVS-11, and integrating them with interactome data using computational and statistical methods. We first determined the differentially expressed genes (DEGs) in each study and horizontally integrated the results at the mRNA and microRNA levels separately. A total of 61 seed genes involved in signal propagation system were obtained by means of unifying mRNA and microRNA detected integrated DEGs. We then reconstructed a refined protein-protein interaction network (PPIN) of infected cells to elucidate the rabies-implicated signal transduction network (RISN). To validate our findings, we confirmed differential expression of randomly selected genes in the network using Real-time PCR. In conclusion, the identification of seed genes and their network neighborhood within the refined PPIN can be useful for demonstrating signaling pathways including interferon circumvent, toward proliferation and survival, and neuropathological clue, explaining the intricate underlying molecular neuropathology of rabies infection and thus rendered a molecular framework for predicting potential drug targets.
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http://dx.doi.org/10.3389/fmicb.2016.01688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5098112PMC
November 2016

Identification of proteins derived from Listeria monocytogenes inducing human dendritic cell maturation.

Tumour Biol 2016 Aug 17;37(8):10893-907. Epub 2016 Feb 17.

Immunology Department, School of Medicine, Tehran University of Medical Sciences, Poursina Avenue, Tehran, Iran.

Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that can promote antitumor immunity when pulsed with tumor antigens and then matured by stimulatory agents. Despite apparent progress in DC-based cancer immunotherapy, some discrepancies were reported in generating potent DCs. Listeria monocytogenes as an intracellular microorganism is able to effectively activate DCs through engaging pattern-recognition receptors (PRRs). This study aimed to find the most potent components derived from L. monocytogenes inducing DC maturation. The preliminary results demonstrated that the ability of protein components is higher than DNA components to promote DC maturation and activation. Protein lysate fractionation demonstrated that fraction 2 HIC (obtained by hydrophobic interaction chromatography) was able to efficiently mature DCs. F2HIC-matured DCs are able to induce allogeneic CD8(+) T cells proliferation better than LPS-matured DCs and induce IFN-γ producing CD8(+) T cells. Mass spectrometry results showed that F2HIC contains 109 proteins. Based on the bioinformatics analysis for these 109 proteins, elongation factor Tu (EF-Tu) could be considered as a PRR ligand for stimulating DC maturation.
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http://dx.doi.org/10.1007/s13277-016-4933-1DOI Listing
August 2016

Designed Amino Acid Feed in Improvement of Production and Quality Targets of a Therapeutic Monoclonal Antibody.

PLoS One 2015 19;10(10):e0140597. Epub 2015 Oct 19.

Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

Cell culture feeds optimization is a critical step in process development of pharmaceutical recombinant protein production. Amino acids are the basic supplements of mammalian cell culture feeds with known effect on their growth promotion and productivity. In this study, we reported the implementation of the Plackett-Burman (PB) multifactorial design to screen the effects of amino acids on the growth promotion and productivity of a Chinese hamster ovary DG-44 (CHO-DG44) cell line producing bevacizumab. After this screening, the amino acid combinations were optimized by the response surface methodology (RSM) to determine the most effective concentration in feeds. Through this strategy, the final monoclonal antibody (mAb) titre was enhanced by 70%, compared to the control group. For this particular cell line, aspartic acid, glutamic acid, arginine and glycine had the highest positive effects on the final mAb titre. Simultaneously, the impact of the designed amino acid feed on some critical quality attributes of bevacizumab was examined in the group with highest productivity. The product was analysed for N-glycan profiles, charge variant distribution, and low molecular weight forms. The results showed that the target product quality has been improved using this feeding strategy. It was shown how this strategy could significantly diminish the time and number of experiments in identifying the most effective amino acids and related concentrations in target product enhancement. This model could be successfully applied to other components of culture media and feeds.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0140597PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4610691PMC
June 2016

Effect of Cysteamine on Cell Growth and IgG4 Production in Recombinant Sp2.0 Cells.

Iran J Pharm Res 2015 ;14(1):177-87

Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

The manipulation of redox potential in secretory pathway by thiol reducing agents can be a strategy to improve the production levels of disulfide-bonded proteins including recombinant antibodies. Here we have studied the influence of cysteamine on viability and the production level of IgG4 in Sp2.0 cells. For this purpose, the recombinant Sp2.0 cells producing an anti CD33 IgG4, were subjected to different concentrations of cysteamine. At concentrations of 2, 4 and 5 mM cysteamine, the secreted levels of IgG4 did not change significantly. However, in concentration of 7 mM cysteamine, a significant decrease was observed in IgG4 levels which may indicate the cytotoxicity of this compound in higher concentrations. Our results show that the cysteamine treatment reduces the cell viability in a dose-dependent manner. Also it was observed that 2 mM cysteamine had no late effect on IgG4 production level and only at day 3, this concentration of cysteamine decreased the cell viability significantly. To test whether the addition of cysteamine can affect the expression level of protein disulfide isomerase, RT-PCR analysis was carried out. The results revealed that cysteamine does not affect the PDI transcription and expression level of IgG4 in this type of recombinant cells.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4277631PMC
January 2015

High efficient expression of a functional humanized single-chain variable fragment (scFv) antibody against CD22 in Pichia pastoris.

Appl Microbiol Biotechnol 2014 Dec 21;98(24):10023-39. Epub 2014 Sep 21.

Biotechnology Research Center, Pasteur Institute of Iran, 13164, Tehran, Iran.

Single-chain variable fragments (scFvs) have recently emerged as attractive candidates in targeted immunotherapy of various malignancies. The anti-CD22 scFv is able to target CD22, on B cell surface and is being considered as a promising molecule in targeted immunotherapy of B cell malignancies. The recombinant anti-CD22 scFv has been successfully expressed in Escherichia coli; however, the insufficient production yield has been a major bottleneck for its therapeutic application. The methylotrophic yeast Pichia pastoris has become a highly popular expression host for the production of a wide variety of recombinant proteins such as antibody fragments. In this study, we used the Pichia expression system to express a humanized scFv antibody against CD22. The full-length humanized scFv gene was codon optimized, cloned into the pPICZαA and expressed in GS115 strain. The maximum production level of the scFv (25 mg/L) were achieved at methanol concentration, 1 %; pH 6.0; inoculum density, OD600 = 3 and the induction time of 72 h. The correlation between scFv gene dosage and expression level was also investigated by real-time PCR, and the results confirmed the presence of such correlation up to five gene copies. Immunofluorescence and flow cytometry studies and Biacore analysis demonstrated binding to CD22 on the surface of human lymphoid cell line Raji and recombinant soluble CD22, respectively. Taken together, the presented data suggest that the Pichia pastoris can be considered as an efficient host for the large-scale production of anti-CD22 scFv as a promising carrier for targeted drug delivery in treatment of CD22(+) B cell malignancies.
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http://dx.doi.org/10.1007/s00253-014-6071-2DOI Listing
December 2014

Comparative proteomics analysis of mice lymphocytes in early stages of infection by different strains of rabies virus.

Indian J Virol 2012 Dec 2;23(3):311-6. Epub 2012 Aug 2.

Protein Chemistry Unit, Biotechnology Research Center, Pasteur Institute of Iran, 69, Pasteur St, 13164 Tehran, Iran.

The CNS immune response to rabies virus has been shown to be influenced by virulence of the virus strains. There is no comprehensive report of the peripheral immune response against different strains of rabies virus. In this report we used a comparative proteome analysis to find the early events in the spleen lymphocytes of mice infected by a street strain and an attenuated strain of the rabies virus. Differentially expressed proteins were identified which play important biological roles such as T and B lymphocyte activation (coronin 1), antiviral activity (peroxiredoxin 1), and cytoskeletal reorganization (cofilin 1). These results could be strong hints of early divergence on peripheral immune response under influence of viral strain and their pathogenicity.
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http://dx.doi.org/10.1007/s13337-012-0093-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3550792PMC
December 2012

Proteomics analysis of human brain tissue infected by street rabies virus.

Mol Biol Rep 2013 Nov 24;40(11):6443-50. Epub 2013 Sep 24.

WHO Collaborating Center for Reference and Research on Rabies, Pasteur Institute of Iran, Tehran, Iran.

In order to extend the knowledge of rabies pathogenesis, a two-dimensional electrophoresis/mass spectrometry based postmortem comparative proteomics analysis was carried out on human brain samples. Alteration in expression profile of several proteins was detected. Proteins related to cytoskeleton, metabolism, proteasome and immune regulatory systems showed the most changes in expression levels. Among these groups, the cytoskeleton related proteins (dynein light chain, β-centractin, tubulin alpha-1C chain and destrin) and metabolism associated proteins (fatty acid-binding protein, macrophage migration inhibitory factor, glutamine synthetase and alpha enolase) were the main altered proteins. These alterations may be considered as an evidence of disturbances in neuronal key processes including axonal transport, synaptic activity, signaling and metabolic pathways in rabies virus infected human brain.
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http://dx.doi.org/10.1007/s11033-013-2759-0DOI Listing
November 2013

The Role of Different Supplements in Expression Level of Monoclonal Antibody against Human CD20.

Avicenna J Med Biotechnol 2013 Jul;5(3):140-7

Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

Background: Recombinant monoclonal antibodies have been marketed in last three decades as the major therapeutic proteins against different cancers. However choosing a proper medium and supplements to reach the high expression is a challenging step. Despite of commercial serum free and chemically defined media, there are still numerous researches seeking the optimum media to gain higher expression titer. Selecting the best basal media followed by proper supplementation to increase the cell density and expression titer needs proper and accurate investigation.

Methods: In this study, we have determined the expression titer of monoclonal antibody against human CD20 using soy extract, Essential Amino Acid, Non-Essential Amino Acid, Panexin NTS, Peptone, Yeast extract, Insulin-transferrin selenite, Human Serum Albumin, Bovine Serum Albumin, Lipid, and two commercially available supplements, Power and Xtreme feed. In each experiment, the expression level was compared with a well defined media, ProCHO5, RPMI 1640 and DMEM-F12.

Results: It has been shown that supplementing the ProCHO5 basal medium with 10% power feed or combination of 5% PanexinNTS,1.5 g/L yeast and 1.5g/L peptone results in the best production levels with 450 and 425 mg/L of anti CD20 mAb expression level, respectively.

Conclusion: Panexin NTS, yeast and peptone cane be proper supplement for fed-batch cell culture instead of commercial Power feed supplement which is a cost effective way to increase expression level. And thereby ProCHO5 may be replaced with common media such as RPMI 1640 and DMEM-F12.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3732863PMC
July 2013

Engineering the cellular protein secretory pathway for enhancement of recombinant tissue plasminogen activator expression in Chinese hamster ovary cells: effects of CERT and XBP1s genes.

J Microbiol Biotechnol 2013 Aug;23(8):1116-22

Biotechnology Research Center, Pasteur Institute of Iran, Tehran 1316943551, Iran.

Cell line development is the most critical and also the most time-consuming step in the production of recombinant therapeutic proteins. In this regard, a variety of vector and cell engineering strategies have been developed for generating high-producing mammalian cells; however, the cell line engineering approach seems to show various results on different recombinant protein producer cells. In order to improve the secretory capacity of a recombinant tissue plasminogen activator (t-PA)-producing Chinese hamster ovary (CHO) cell line, we developed cell line engineering approaches based on the ceramide transfer protein (CERT) and X-box binding protein 1 (XBP1) genes. For this purpose, CERT S132A, a mutant form of CERT that is resistant to phosphorylation, and XBP1s were overexpressed in a recombinant t-PA-producing CHO cell line. Overexpression of CERT S132A increased the specific productivity of t-PA-producing CHO cells up to 35%. In contrast, the heterologous expression of XBP1s did not affect the t-PA expression rate. Our results suggest that CERTS132A- based secretion engineering could be an effective strategy for enhancing recombinant t- PA production in CHO cells.
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http://dx.doi.org/10.4014/jmb.1302.02035DOI Listing
August 2013

Neutralizing antibodies in multiple sclerosis patients on weekly intramuscular Avonex and biosimilar interferon beta-1a (CinnoVex): comparing results of measurements in two different laboratories.

J Immunol Methods 2013 Feb 6;388(1-2):46-8. Epub 2012 Dec 6.

Research Coordinator, Isfahan University of Medical Sciences, Isfahan, Iran.

The appearance of neutralizing antibodies (NAbs) has significant clinical and regulatory consequences for interferons in patients with multiple sclerosis (MS). In a double blind, randomized clinical trial, 84 patients with relapsing remitting MS were enrolled in a 24month study period. Patients were randomly assigned into two groups receiving 30mcg weekly intramuscular injections of either Avonex® (Biogen Idec, USA; 42 patients) or CinnoVex® (CinnaGen Co, Iran; 42 patients). NAb titer was drawn for all patients every 6months and assayed using cytopathic effect assay (CPE) method in Tehran, Iran. To validate the measure done in the Iranian lab, 45 sera with adequate volume and proper storing condition were selected and sent to be rechecked using luciferase reporter gene assay (LA) method for verification in 2 phases in Vancouver, Canada. The cut-off point of 20 TRU was considered for positivity. The two labs found the same three samples to be positive (2 samples from patients received Avonex and 1 received CinnoVex) and 42 to be negative. They had the following values using the Kawade formula as recommended by international standards; 2238, 89 and 302 (TRu/ml) using CPE assay versus 2464, 290 and 169 (TRu/ml) using LA method. As similar results were obtained from CinnoVex or Avonex in our study, we suggest that both medications will have a similar immunogenetic profile.
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http://dx.doi.org/10.1016/j.jim.2012.11.013DOI Listing
February 2013

Expression changes of cytoskeletal associated proteins in proteomic profiling of neuroblastoma cells infected with different strains of rabies virus.

J Med Virol 2013 Feb 20;85(2):336-47. Epub 2012 Nov 20.

Protein Chemistry Unit, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

Rabies virus invades the nervous system, induces neuronal dysfunction and causes death of the host. The disruption of the cytoskeletal integrity and synaptic structures of the neurons by rabies virus has been postulated as a possible basis for neuronal dysfunction. In the present study, a two-dimensional electrophoresis/mass spectrometry proteomics analysis of neuroblastoma cells revealed a significant effect of a virulent strain of rabies virus on the host cytoskeleton related proteins which was quite different from that of an attenuated strain. Vimentin, actin cytoplasmic 1 isoform, profilin I, and Rho-GDP dissociation inhibitor were host cell cytoskeletal related proteins changed by the virulent strain. The proteomics data indicated that the virulent strain of rabies virus induces significant expression changes in the vimentin and actin cytoskeleton networks of neurons which could be a strong clue for the relation of cytoskeletal integrity distraction and rabies virus pathogenesis. In addition, the expression alteration of other host proteins, particularly some structural and regulatory proteins may have potential roles in rabies virus pathogenesis.
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http://dx.doi.org/10.1002/jmv.23458DOI Listing
February 2013

Annexin C4 in A. fumigatus: a proteomics approach to understand the function.

J Proteomics 2011 Sep 27;74(10):1950-8. Epub 2011 May 27.

Fungal Biotechnology Group, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

Annexin C4 has been identified as a new member of fungal annexin family. In search of function, we have generated an annexin C4 disruptant strain of human pathogen, Aspergillus fumigatus. Detailed phenotypic analysis confirmed a non essential role of annexin C4 in the growth and sporulation of this pathogen. We applied a comparative proteomics strategy to understand the possible role of this protein in the fungus. The modification of respiratory chain proteins and stress response proteins suggests the occurrence of a mild oxidative stress in anxC4 disruptant strain. This may indicate a possible anti stress function of annexin C4 in A. fumigatus.
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http://dx.doi.org/10.1016/j.jprot.2011.05.018DOI Listing
September 2011

Potential molecular targets in chemopreventative action of celecoxib: a proteomics analysis of J774.A1 macrophage-like cell line.

Mol Biosyst 2011 Apr 24;7(4):1306-11. Epub 2011 Jan 24.

Toxicology and Pharmacology Department, Faculty of Pharmacy, Tehran University of Medical Sciences, Iran.

The overexpression of cyclooxygenase-2 (COX-2) enzyme has been strongly contributed to tumorigenesis. The efficacy of celecoxib as a selective COX-2 inhibitor has been shown in many studies, but the underlying mechanism as a chemopreventative agent has not yet been well known. For better understanding the chemopreventative molecular mechanisms, we used a comparative proteomics analysis of lipopolysaccharide (LPS) treated and untreated J774.A1 macrophage-like cell lines before and after treatment with celecoxib. Our findings define the contribution of several interesting proteins, including ferritin heavy chain, glyoxalase-1, cofilin, vimentin, and galectin-1, which could extend our understanding of the chemopreventative effects of celecoxib and provide new valuable tools for further anticancer research.
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http://dx.doi.org/10.1039/c0mb00201aDOI Listing
April 2011

Fluorescent Leishmania species: development of stable GFP expression and its application for in vitro and in vivo studies.

Exp Parasitol 2011 Mar 25;127(3):637-45. Epub 2010 Dec 25.

Molecular Immunology and Vaccine Research Laboratory, Pasteur Institute of Iran, Tehran, Iran.

Reporter genes have proved to be an excellent tool for studying disease progression. Recently, the green fluorescent protein (GFP) ability to quantitatively monitor gene expression has been demonstrated in different organisms. This report describes the use of Leishmania tarentolae (L. tarentolae) expression system (LEXSY) for high and stable levels of GFP production in different Leishmania species including L. tarentolae, L. major and L. infantum. The DNA expression cassette (pLEXSY-EGFP) was integrated into the chromosomal ssu locus of Leishmania strains through homologous recombination. Fluorescent microscopic image showed that GFP transgenes can be abundantly and stably expressed in promastigote and amastigote stages of parasites. Furthermore, flow cytometry analysis indicated a clear quantitative distinction between wild type and transgenic Leishmania strains at both promastigote and amastigote forms. Our data showed that the footpad lesions with GFP-transfected L. major are progressive over time by using fluorescence small-animal imaging system. Consequently, the utilization of stable GFP-transfected Leishmania species will be appropriate for in vitro and in vivo screening of anti-leishmanial drugs and vaccine development as well as understanding the biology of the host-parasite interactions at the cellular level.
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http://dx.doi.org/10.1016/j.exppara.2010.12.006DOI Listing
March 2011

Increased expression of recombinant human tissue plasminogen activator in Leishmania tarentolae.

Biotechnol J 2010 Nov;5(11):1198-206

Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

Recombinant tissue plasminogen activator (rt-PA) is one of the most important thrombolytic agents for treating cardiovascular obstructions such as stroke. Glycoprotein rt-PA is a serine protease, consisting of 527 amino acids of which 35 are cysteine residues. A variety of recombinant protein expression systems have been developed for heterologous gene expression in prokaryotic and eukaryotic hosts. In recent years, Leishmania tarentolae has been considered because of its safety aspects and special attributes in expression of complex proteins. In this study, two expression cassettes, each one including two copies of t-PA cDNA, were used for integration into the L. tarentolae genome by electroporation. Transformed clones were selected in the presence of appropriate antibiotics. Expression of active rt-PA was confirmed by Western blot and Zymography tests. Real-time PCR analysis was applied to investigate the presence of multiple t-PA gene copies in the parasite genome. Correlation of t-PA gene dosage and production rate was confirmed with real-time PCR. It was shown that the expression level of rt-PA in L. tarentolae is at least 480 IU/mL of culture media. This concentration of rt-PA is seven times higher than what was reported in previous studies in L. tarentolae and some other eukaryotic systems.
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http://dx.doi.org/10.1002/biot.201000233DOI Listing
November 2010