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Background: Overexpression of CD20 protein on the surface of B cells in lymphoma can be targeted by several anti-CD20 molecules. The development of accessible interactive epitopes is more favorable than the full-length transmembrane CD20 in the affinity assessment of anti-CD20 monoclonal antibodies (mAbs). Methods: The sequence of these epitopes was extracted, and the effects of different linker peptides and the location of histidine (His)-tag were computationally analyzed. The impact of thioredoxin (Trx)-tag on the folding of the selected construct and its interaction with rituximab was further investigated. The two final expression cassettes were expressed in Escherichia coli after optimization of culture conditions for incubation temperature, post-induction time, optical density at the induction time, and concentration of the inducer. ELISA evaluated the binding affinity of rituximab towards the recombinant proteins. Results: By homology modeling studies, C-terminal His-tagged structures represented more desirable folded structures. Validation of the models revealed that CD20 extracellular domain linked by the G4S polypeptide had better stereochemical quality and structural compatibility. It was selected due to its more effective interaction with rituximab showing the highest dissociation constant of 5.8E-09M, which improved after the fusion of Trx-tag (7.1E-10M). The most influential parameters in the expression of the two selected proteins were post-induction temperature and optical density at the induction time. Homemade ELISA assays revealed a slightly higher affinity of rituximab towards the Trx-CD20 protein than the CD20/G4S molecule. Conclusions: Experimental in vitro studies confirmed the computationally calculated affinity of rituximab towards the two designed CD20 constructs. Also, the cell-based binding assessment of anti-CD20 mAbs could be substituted by the engineered extracellular domain of human CD20 protein.

Background Bispecific antibodies represent an important class of monoclonal antibodies (mAbs), with great therapeutic potentials due to their ability to target simultaneously two distinct epitopes. The generation of functional bispecific antibodies with the highest possible yields is particularly critical for the production of these compounds on industrial scales. Anti-CD3 × CD19 bispecific antibody (bsAb) is a bispecific T-cell engager currently used for treating ALL. Herein, we have tried to optimize the expression level of this antibody in mammalian hosts. Methods woodchuck hepatitis virus post-transcriptional regulation (WPRE) sequence was incorporated at the 3' end of the expression cassette. This modification resulted in a notable about two-fold increase in the expression of the bsAb in the Expi293 cell line. Results & conclusion Follow-up flow cytometry analysis demonstrated the binding properties of the produced antibody at acceptable levels, and in vitro bioactivity assays showed that this product is potent enough for targeting and destroying CD19-positive cells. Our findings show that WPRE enhances the expression of this type of bispecific mAbs in human embryonic kidney-293 family cell lines. This approach can be used in biopharma industry for the mass production of anti-CD3 × CD19 bispecific antibody.

With the increasing dominance of monoclonal antibodies (mAbs) in the biopharmaceutical industry and smaller antibody fragments bringing notable advantages over full-length antibodies, it is of considerable significance to choose the most suitable production system. Although mammalian expression system has been the preferred choice in recent years for mAbs production, E. coli could be the favorable host for non-glycosylated small antibody fragments due to the emergence of new engineered E. coli strains capable of forming disulfide-bonds in their cytoplasm. In this study, non-glycosylated anti-TNF-α Fab’ moiety of Certolizumab pegol, produced by periplasmic expression in E. coli in previous studies, was produced in the cytoplasm of E. coli SHuffle strain. The results indicated that it is biologically functional by testing the antigen-binding activity via indirect ELISA and inhibition of TNF-α induced cytotoxicity using MTT test. Major factors affecting protein production and, optimized culture conditions were examined by analyzing growth characteristics and patterns of expression in 24 h of post-induction cultivation and, optimization of culture conditions by response surface methodology considering temperature, time of induction and concentration of inducer in small (tube) and shake-flask scale. Based on the results, temperature had the most significant influence on functional protein yield while exerting different impacts in small and shake-flask scales, which indicated that cultivation volume is also an important factor that should be taken into account in optimization process. Furthermore, richness of medium and slower cellular growth rate improved specific cellular yield of functional protein by having a positive effect on the solubility of Fab' antibody.

Background Multiple sclerosis (MS) is a multifactorial condition in which many genetic and environmental factors interfere. The association between genes involved in the immune system and MS was previously reported. The aims of this study were to evaluate 14 SNPs of HLA-DRA, 14 SNPs of IL2RA with severity of MS through Expanded Disability Status Scale (EDSS) and Annualized Relapse Rate (ARR). Methods 102 patients with MS referred to Sina hospital in Tehran, Iran, were diagnosed and studied based on McDonald's guideline, clinical signs, and brain imaging procedures. All patients were included in the study following informed consent. Genotyping study of 14 variants in the HLA-DRA, and 14 variants in IL2RA was conducted by Sanger sequencing. Disease outcomes including EDSS and ARR were registered. Outcome measures between different genotypes of each SNPs were compared separately. Results Among 14 SNPs in IL 2RA the genotypes of rs12722489 showed a significant association with ARR in two consecutive years. Mean ARR1 was 1.06±1.12, 0.20±0.34 and 0.31±.50 for AA, GA, and GG genotypes, respectively (p value= 0.008). Mean ARR2 was 1.5±1.08, 0.28±0.40, and 0.42±0.55 for AA, GA, and GG, respectively (p value= 0.001). Regression analysis showed a significant association between rs12722489 with ARR1 and ARR2, removing the potential confounding mediators. No significant association was found between SNPs in HLA-DRA with the attack rate and severity of MS. Conclusion The rs12722489 of IL-2RA has an association with ARR, but not with EDSS.

Background Multiple sclerosis (MS) is a common demyelinating neurodegenerative disorder with significant heritability. Previous studies have associated genetic variants in human leukocyte antigen (HLA) complex, IL2RA , and HMGB1 genes with the pathophysiology of MS. Methods In order to investigate the gene association in the Iranian population, we performed a genotyping study of 36 variants in the mentioned genes using Sanger sequencing in 102 MS patients and 113 healthy controls. Results Our results identified significant associations as well as significant allele frequency differences in some of the studied single-nucleotide polymorphisms including rs4935356, rs3177928, and rs7197 from HLA-DRA gene, and rs12722489 and rs12722490 variants from IL2RA gene (p< 0.05). Moreover, the strong linkage disequilibrium of two common haplotypes was estimated from the HLA-DRA gene. Conclusion This association study may suggest the role of these polymorphisms in the genetic susceptibility of MS in the Iranian population and would facilitate the recognition of causative variants in this disease.

Background:Protein misfolding is a common problem in large-scale production of recombinant proteins, which can significantly reduce the yield of the process.Objective:In this work, we aimed at treating a cell culture broth containing high levels (>45%) of incorrectly folded Fc-fusion proteins by a simple redox buffer system in order to increase the proportion of the protein with correct conformation.Methods:Multi-variable process optimization was firstly conducted at a small scale (25 mL), employing an experimental design methodology. After identifying the key variables using a resolution IV Fractional Factorial Design (FFD), the process was then optimized by the Central Composite Design (CCD).Results:The optimal conditions for the refolding reaction were 340 mM Tris-base, 6.0 mM L-cysteine, 0.5 mM L-cystine, a buffer pH of 9.0, a reaction temperature of 8.5ºC and a reaction time of 24 h. Based on the treatment conditions obtained at a small scale, the process was further scaled up to 4500- L. The misfolded content was always less than 20%. The reaction can proceed well in the absence of chemical additives, such as chaotropic agents, aggregation suppressors, stabilizers and chelators.Conclusion:The refolding process increases the fraction of active protein in the original broth reducing the burden on downstream purification steps markedly.

BACKGROUND Multiple sclerosis (MS) is a chronic disease with significant morbidity. A wide spectrum of risk factors has been suggested that triggers the development of MS. Among them, several viral infections have been implicated to play a role in MS pathogenesis. We aimed to evaluate the relationship between viral diseases, including Epstein–Barr virus (EBV), human herpes virus 6 (HHV-6), cytomegalovirus (CMV), and hepatitis B virus (HBV) and MS in the present case-control study. METHODS About 100 patients with confirmed MS and age- and sex-matched individuals were selected as case and control groups, respectively. The patients were randomly selected from individuals diagnosed by neurologists based on the clinical signs and symptoms and imaging procedures. RESULTS More than 100 patients with MS and patients who were referred for other causes were analyzed for the presence of DNA of EBV, HHV6, CMV, and HBV separately. 9.37% of the control group had a positive test for the DNA of EBV in a real-time polymerase chain reaction (PCR), while the frequency of positive test result was zero in the case group (p = 0.0012). HBV DNA was not detected in both the case and control groups. The prevalence of CMV was 0.88 and zero in the control and case groups, respectively (p = 0.3410). For HHV6, 9.73 % of the control group had a positive result, while this test was positive in 5.88% of the patients with MS (p = 0.2959). CONCLUSION We detected a significantly higher number of individuals with DNA of EBV in their blood among the control group compared with the case group. In conclusion, the results suggest a surprisingly adverse association between MS and EBV, and no association was found between the presence of DNA of HBV, CMV, and HHV6 and MS.

One of the important interactions in controlling the human immune system is the reaction between checkpoint proteins such as programmed cell death-1 (PD-1) and its ligand, PD-L1. These are negative immunoregulatory molecules that promote immune evasion of tumor cells. PD-L1 expression is an immune-mediated mechanism used by various malignant cells in order to down-regulate the immune system. Checkpoint inhibitors (CPIs) are a new class of anti-cancer agents that stimulate immune cells to elicit an antitumor response by blocking the ligand and receptor interactions. Nanobody (Nb) as a new type of antibody fragment, has some potential as CPI.A female camel was immunized with recombinant PD-L1 protein, nanobody library was constructed and PD-L1 specific Nb was selected. The selected Nb was characterized in terms of affinity, specificity, and binding potency in ELISA, Western blotting, and flow cytometry.Developed nanobody, A22 binds to its cognate target with high specificity and affinity. Western blot and flow cytometry techniques showed that nanobody A22 was able to specifically detect and attach to human PD-L1 protein on the cell surface and in the cell lysate. MTT assay showed the inhibitory effect of PD-L1 by specific Nb on A431 and HEK293 cells, with no cytotoxic effect on cell growth.The results highlighted the potential of anti-PD-L1 Nb as a novel therapeutic in cancer therapy without undesirable cytotoxicity.

It is widely accepted that the growing demand for recombinant therapeutic proteins has led to the expansion of the biopharmaceutical industry and the development of strategies to increase recombinant protein production in mammalian cell lines such as SP2/0 HEK and particularly Chinese hamster ovary cells. For a long time now, most investigations have been focused on increasing host cell productivity using genetic manipulating of cellular processes like cell cycle, apoptosis, cell growth, protein secretory and other pathways. In recent decades MicroRNAs beside different genetic engineering tools (e.g., TALEN, ZFN, and Crisper/Cas) have attracted further attention as a tool in the genetic engineering of host cells to increase protein expression levels. Their ability to simultaneously target multiple mRNAs involved in one or more cellular processes made them a favorable tool in this field. Accordingly, this study aimed to review the methods of selecting target miRNA for cell line engineering, miRNA gain- or loss-of-function strategies, examples of laboratory and pilot studies in this field and discussed advantages and disadvantages of this technology.

Although urine cytology and cystoscopy are current gold standard methods in diagnosis and surveillance of Bladder cancer (BC), they have some limitations which necessitates novel diagnostic approaches to compensate their drawbacks. In this regard, Nuclear Matrix Protein 22 (NMP22) is introduced as a potential tumor biomarker for BC detection (FDA approved). NMP22 determination mainly occurs through immunoassay platforms, raising a proper antibody against its antigen. Hence, development of such immunoassays seems crucial. Various bioinformatic tools were harnessed to select a region with lowest variability, highest density for linear and conformational epitopes, lowest post translational modifications, highest antigenicity, best physicochemical properties and reliable transcriptional properties. Subsequently, E. coli BL21 (DE3) and P. pastoris GS115 were applied for exogenous expression. Ultimately, protein purification and quantification was followed by ELISA test for antibody analyses. Both host successfully expressed the antigen, while the E. coli expression was with higher yield. The commercial anti-NMP22 antibodies showed relatively equal detection results. However, the slight better detection for the antigen with P. pastoris origin could be deduced as better structural properties for P. pastoris. These results indicate higher expression yields and lower costs for over-expression of this eukaryotic antigen.

Secreted recombinant activated clotting factor VII activated (rFVIIa) in cell culture media missing gamma-carboxyglutamic acid (Gla) domain as a result of failure in gamma-carboxylation or cell lysis is called Gla-domainless impurity which has less negative charge compared to native rFVIIa. Based on risk assessment, this type of impurity is considered as critical drug product quality attribute of rFVIIa and its quantitative analysis in product batches is a critical issue in quality control laboratories. Analysis of Gla-domainless impurity is accomplished by Strong Anion Exchange Chromatography (SAX) in recombinant factor VIIa using Tris and Bis-Tris propane salt buffers as equilibrating buffers and high concentration ammonium acetate as an eluent. Appearance of ghost peaks with notable intensity during elution time of Gla-domainless impurity caused distortion of the related peak and interference with robust and accurate quantification of this impurity. Subsequently, the ghost peak was analyzed by LC-ESI-MS to determine the structure which showed the m/z values at 905.27, 623.53 and 341.60 and 563.73. To find the source of these ghost peaks, quality of water, buffer salts and Chelex-100 together with ionic strength of mobile phase A (addition of 25 mM NaCl) were considered as affecting parameters and several experiments designed with DOE software to optimize the best condition of highest quality the method with lowest signal of ghost peak noises. By interpretation of DOE result, it is concluded that high grade water and buffer salt along with high quality Chelex-100 resins are important factors to achieve a method with lowest ghost peaks. However, addition of 25 mM NaCl to mobile phase A with either lower quality buffer salts or lower water grade yields high quality chromatogram peak with acceptable ghost peaks. LC/MS analysis indicates that macrostructures of Bis-Tris propane made up as a result of hydrogen bonds with each other or Tris molecules can be the source of ghost peaks.

In recent decades, immunotoxins have attracted significant attention in treatment of a wide range of diseases including cancers due to their natural origins and their role in blocking crucial pathways within the cells. Ribosome inactivating proteins (RIPs) are efficient molecules in blocking protein synthesis through interactions with ribosomal rRNA molecules. cDNA molecule encoding HER2 scFv antibody fragment originated from trastuzumab attached to the mature alpha luffin gene fragment was subcloned into pET28a expression vector and expressed in different E. coli expression hosts. Identity of the expressed recombinant protein was investigated through western blotting and the fusion protein was purified using Ni-NTA affinity chromatography. The biological activity (toxicity) of the protein was investigated on DNA and RNA samples. A 58 kDa protein was expressed in E. coli. The best protein expression level was achieved in 0.2 mM IPTG at 30 °C in TB medium using E. coli BL21 (DE3) host strain. The fusion protein showed RNase and DNA glycosylase activity on tested RNA and DNA samples. DNA glycosylase activity of the recombinant fusion protein showed that alpha luffin part of this protein is active in conjugation to the scFv molecule and the expressed protein can be further studied in targeted biological in vitro assays.

Despite the success of monoclonal antibodies (mAbs) to treat some disorders, the monospecific molecular entity of mAbs as well as the presence of multiple factors and pathways involved in the pathogenesis of disorders, such as various malignancies, infectious diseases, and autoimmune disorders, and resistance to therapy have restricted the therapeutic efficacy of mAbs in clinical use. Bispecific antibodies (bsAbs), by concurrently recognizing two targets, can partly circumvent these problems. Serial killing of tumor cells by bsAb-redirected T cells, simultaneous blocking of two antigens involved in the HIV-1 infection, and concurrent targeting of the activating and inhibitory receptors on B cells to modulate autoimmunity are part of the capabilities of bsAbs. After designing and developing a large number of bsAbs for years, catumaxomab, a full-length bsAb targeting EpCAM and CD3, was approved in 2009 to treat EpCAM-positive carcinomas besides blinatumomab, a bispecific T cell engager antibody targeting CD19 and CD3, which was approved in 2014 to treat relapsed or refractory acute lymphoblastic leukemia. Furthermore, approximately 60 bsAbs are under investigation in clinical trials. The current review aims at portraying different formats of the single-chain variable fragment (scFv)-based bsAbs and shedding light on the scFv-based bsAbs in preclinical development, different phases of clinical trials, and the market. Keywords: monoclonal antibody, single-chain variable fragment, bispecific antibody, cancer, HIV-1, bacteria, ESKAPE, autoimmune diseases

The cationic polyelectrolyte pDADMAC is widely used in biopharmaceutical industry as a flocculating agent to enhance clarification throughput and downstream filtration operations. Due to the possible toxicity, pDADMAC should be assessed for an acceptable residual level to ascertain the safety of the product to patients. The strong protein-polyelectrolyte interaction, however, can negatively affect sensitivity and accuracy of measurements. This paper reports on the application of size exclusion (SE) chromatography coupled to evaporative light scattering detector (ELSD) to the quantitative determination of pDADMAC in monoclonal antibody formulations and in process intermediates during downstream purification. The SE chromatography was performed under isocratic condition with a mobile phase consisting of 0.1% TFA in water (90%) and acetonitrile (10%) at a flow rate of 0.4 ml/min. A quantification limit (S/N = 10) of 0.85 ppm was achieved in sample matrix, which is sufficiently low for the trace analysis of this compound in protein-containing samples.

Dareshouri, Arabian, and Akhal-Teke are 3 Middle Eastern horse breeds that have been selected for endurance and adaptation to harsh climates. Deciphering the genetic characteristics of these horses by tracing selection footprints and copy number of variations will be helpful in improving our understanding of equine breeds’ development and adaptation. For this purpose, we sequenced the whole genome of 4 Dareshouri horses using Illumina Hiseq panels and compared them with publicly available whole-genome sequences of Arabian (n = 3) and Akhal-Teke (n = 3) horses. Three tests of FLK, hapFLK, and pooled heterozygosity were applied using a sliding window (window size = 100 kb, step size = 50 kb) approach to detect putative selection signals. Copy number variation analysis was applied to investigate copy number of variants (CNVs), and the results were used to suggest selection signatures involving CNVs. Whole-genome sequencing demonstrated 8 837 950 single-nucleotide polymorphisms (SNPs) in autosomal chromosomes. We suggested 58 genes and 3 quantitative trait loci, including some related to horse gait, insect bite hypersensitivity, and withers height, based on selective signals detected by adjusted P-value of Mahalanobis distance based on the rank-based P-values (Md-rank-P) method. We proposed 12 genomic regions under selection pressure involving CNVs that were previously reported to be associated with metabolism energy (SLC5A8), champagne dilution in horses (SLC36A1), and synthesis of polyunsaturated fatty acids (FAT2). Only 10 Middle Eastern horses were tested in this study; therefore, the conclusions are speculative. Our findings are useful to better understanding the evolution and adaptation of Middle Eastern horse breeds.

BackgroundSHuffle is a suitableEscherichia coli(E. coli) strain for high yield cytoplasmic soluble expression of disulfide-bonded proteins such as Insulin due to its oxidative cytoplasmic condition and the ability to correct the arrangement of disulfide bonds. Lispro is an Insulin analog that is conventionally produced inE. colias inclusion bodies (IBs) with prolonged production time and low recovery. Here in this study, we aimed to optimize cultivation media composition for high cell density fermentation of SHuffle T7E. coliexpressing soluble Lispro proinsulin fused to SUMO tag (SU-INS construct) to obtain high cell density fermentation.ResultsFactors including carbon and nitrogen sources, salts, metal ions, and pH were screened via Plackett–Burman design for their effectiveness on cell dry weight (CDW) as a measure of cell growth. The most significant variables of the screening experiment were Yeast extract and MgCl2concentration, as well as pH. Succeedingly, The Central Composite Design was utilized to further evaluate and optimize the level of significant variables. The Optimized media (OM-I) enhanced biomass by 2.3 fold in the shake flask (2.5 g/L CDW) that reached 6.45 g/L (2.6 fold increase) when applied in batch culture fermentation. The efficacy of OM-I media for soluble expression was confirmed in both shake flask and fermentor.ConclusionThe proposed media was suitable for high cell density fermentation ofE. coliSHuffle T7 and was applicable for high yield soluble expression of Lispro proinsulin.

Anti-TNF inhibitors exert their therapeutic effect by inhibition of the excessive amounts of TNF-α within the body. Recombinant TNF-α should be produced in a soluble refolded form to investigate the effectiveness and efficiency of anti-TNF-α compounds. In this research, the designed cassette was subcloned in the pET28a expression vector and expressed in E. coli BL21 (DE3). The identity of the protein was confirmed through SDS-PAGE and Western blotting. After optimizing expression conditions, protein purification was performed using native Ni-NTA affinity chromatography. The biological activity of the soluble recombinant TNF-α was investigated using MTT assay. Also, the affinity of an anti-TNF-α agent, Altebrel, was investigated against the expressed protein through ELISA. Optimization of TNF-α expression conditions represented that the highest expression could be achieved at 37 °C using 0.5 mM IPTG 6 h post-induction. The recombinant protein represented an inhibitory effect on the L929 murine fibroblast cell line and was successfully detected by Altebrel in ELISA. Binding kinetics were also studied using Cimzia as an anti-TNF-α molecule and 7.2 E−13M was calculated as the equilibrium dissociation constant value (KD). The significant expression level of the recombinant protein in the soluble form, its high purity, and assessment of its biological activity showed that the expressed protein could be used in tests of ELISA and MTT to assess the activity of anti-TNF-α agents.

BACKGROUND: Multiple sclerosis (MS) is a common demyelinating neurodegenerative disorder with significant heritability. Previous studies have associated genetic variants in human leukocyte antigen (HLA) complex, IL2RA , and HMGB1 genes with the pathophysiology of MS. METHODS: In order to investigate the gene association in the Iranian population, we performed a genotyping study of 36 variants in the mentioned genes using Sanger sequencing in 102 MS patients and 113 healthy controls. RESULTS: Our results identified significant associations as well as significant allele frequency differences in some of the studied single-nucleotide polymorphisms including rs4935356, rs3177928, and rs7197 from HLA-DRA gene, and rs12722489 and rs12722490 variants from IL2RA gene (p< 0.05). Moreover, the strong linkage disequilibrium of two common haplotypes was estimated from the HLA-DRA gene. CONCLUSION: This association study may suggest the role of these polymorphisms in the genetic susceptibility of MS in the Iranian population and would facilitate the recognition of causative variants in this disease.

BACKGROUND: Multiple sclerosis (MS) is a multifactorial condition in which many genetic and environmental factors interfere. The association between genes involved in the immune system and MS was previously reported. The aims of this study were to evaluate 14 SNPs of HLA-DRA, 14 SNPs of IL2RA with severity of MS through Expanded Disability Status Scale (EDSS) and Annualized Relapse Rate (ARR). METHODS: 102 patients with MS referred to Sina hospital in Tehran, Iran, were diagnosed and studied based on McDonald’s guideline, clinical signs, and brain imaging procedures. All patients were included in the study following informed consent. Genotyping study of 14 variants in the HLA-DRA, and 14 variants in IL2RA was conducted by Sanger sequencing. Disease outcomes including EDSS and ARR were registered. Outcome measures between different genotypes of each SNPs were compared separately. RESULTS: Among 14 SNPs in IL 2RA the genotypes of rs12722489 showed a significant association with ARR in two consecutive years. Mean ARR1 was 1.06±1.12, 0.20±0.34 and 0.31±.50 for AA, GA, and GG genotypes, respectively (p value= 0.008). Mean ARR2 was 1.5±1.08, 0.28±0.40, and 0.42±0.55 for AA, GA, and GG, respectively (p value= 0.001). Regression analysis showed a significant association between rs12722489 with ARR1 and ARR2, removing the potential confounding mediators. No significant association was found between SNPs in HLA-DRA with the attack rate and severity of MS. CONCLUSION: The rs12722489 of IL-2RA has an association with ARR, but not with EDSS.

Certolizumab pegol is a Fab′ antibody fragment for treatment of rheumatoid arthritis and Crohn’s disease which is conjugated to a 40 kDa PEG molecule in order to increase the protein half-life. PEGylation may have disadvantages including immunogenicity, hypersensitivity, vacuolation, decreased binding affinity and biological activity of the protein. To overcome these problems, PASylation has been developed as a new approach. The nucleotide sequence encoding 400 amino acid PAS residues was genetically fused to the corresponding nucleotide sequences of both chains of certolizumab. Then, the bioactivity as well as physicochemical and pharmacokinetic properties of the recombinant PASylated expressed protein was assayed. Circular dichroism spectroscopy demonstrated that the random coil structure of PAS sequences did not change the secondary structure of the PASylated Fab′ molecule. It was observed that PASylation influenced the properties of the Fab′ molecule by which the hydrodynamic radius and neutralization activity were increased. Also, the antigen binding and binding kinetic parameters improved in comparison to the PEGylated Fab′ antibody. Pharmacokinetic studies also showed prolonged terminal half-life and improved pharmacokinetic parameters in PASylated recombinant protein in comparison to the PEGylated and Fab′ control molecules. The results reconfirmed the efficiency of PASylation approach as a potential alternative method in increasing the half-life of pharmaceutical proteins.

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 in vitro 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 in vitro functional similarity between Altebrel™ and Enbrel®.

This study is to investigate the binding ability of Designed Ankyrin Repeat Proteins type Ec1that was fused to Low Molecular Weight Protamine (DARPin Ec1-LMWP) protein scaffold to the Epithelial Cell Adhesion Molecule (EpCAM) Cancer Stem Cell (CSC) marker and its efficiency in targeted delivery of small interfering RNA (siRNA) molecules into the studied cells. Gene fragment encoding the DARPIn Ec1-LMWP fusion protein was subcloned into pET28a expression vector following molecular docking studies performed to examine the affinity of the fusion protein towards EpCAM marker. The binding of the siRNA to the expressed fusion protein was tested through native PAGE. The toxicity of the fusion protein was tested by MTT assay. Attachment of the complex to the EpCAM marker was investigated by flow cytometry and delivery of siRNA into the cells was assessed by fluorescence microscopy. The expressed 21.6 kDa DARPin Ec1-LMWP fusion protein was purified and showed no cytotoxicity on tested cells. Arginine rich LMWP was efficiently bounded to the negatively charged siRNA molecule. Successful attachment of the fusion protein:siRNA complex to the EpCAM marker and its internalization into MCF-7 breast cancer cell line were confirmed. Here for the first time the recombinant DARPin Ec1-LMWP protein scaffold was designed and tested for targeting EpCAM surface marker and successful internalization of the siRNA into MCF-7 cells.

The "bispecifics" market improved over the past decade due to the development of many technological platforms including bispecific T cell engagers (BiTEs). The approval of blinatumomab, the most advanced bispecific T-cell engager (BiTE) in clinical trials, can be a significant milestone in the development of bispecific antibodies. Both Chinese hamster ovary (CHO) cells and E. coli strain are considered as the most widely used hosts for the large-scale production of therapeutic monoclonal antibodies. Since both of the economic and qualitative aspects of protein production are important in industry, selection of a suitable protein expression system is very critical. The BsAb gene was cloned into the expression vectors FC550A-1, pcDNA3.1 (+), and PET22b and 6 × His-tagged BsAb then purified on a Ni-NTA chromatography column. Both SDS-PAGE and Western blotting analysis of the purified protein demonstrated that blinatumomab was successfully expressed as a 55 kDa in both expression systems. The antigen-binding properties of blinatumomab were compared in the mammalian system versus Escherichia coli. The results showed that the purified antibody from a mammalian expression system has better binding activity than the one from E. coli host.

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.

Etanercept is a soluble fusion protein of the tumor necrosis factor receptor (TNFR) extracellular domain, linked to an Fc part of IgG1. It possesses three N- and 13 O-glycosylation sites. Due to its complex structure, an analytical challenge is facing the development and approval of biosimilars. In the current study, physicochemical characterization using state-of-the-art analytics was performed to analyze intact and subunit masses, post-translational modifications (PTMs), higher order structure and potency of Etanercept originator Enbrel® and its biosimilar Altebrel™ (AryoGen Pharmed) in accordance to critical quality attributes of biopharmaceuticals. Intact mass and subunit analysis revealed a size of about 126 kDa for both biologicals. Similar glycoprotein species for the complete monomer and the Fc domain of originator and follow-on product were observed, however, small differences in lysine variants and oxidation were found. N-Glycopeptide analysis with UHPLC-QTOF-MSE confirmed the N-glycosylation sites (N149, N171 and N317) as well as Fc-specific glycosylation on N317, and TNFR-specific highly sialylated glycans on N149 and N171 on both investigated products. Small quantitative variations in the N-glycan profile were detected, although the N-glycans were qualitatively similar. Four different O-glycopeptides bearing core 1-type glycans were detected. For both, N- and O-glycopeptide analysis, determination was achieved without prior cleavage of the sialic acid residues for the first time. In addition, ion mobility spectrometry data confirmed close similarity of higher-order structure of both biologics. Furthermore, a neutralization assay, investigating the impact of altered PTMs on potency, indicated that the differences within all batches are still in the acceptable range for biosimilarity.

An effective Leishmania vaccine must induce strong Th1 immune responses and generate long-term effectiveness in the host. To date, most designated anti-Leishmania vaccines have shown poor efficacy in clinical studies. One of the most effective approaches in enhancing the efficacy of vaccines to generate protective immunity is the use of multiple distinct antigens in a single vaccination regimen. For this reason, we investigated the immunogenicity and protective efficacy of liposomal formulation composed of DOTAP:Cholesterol, protamine and CpG ODNs (LPD NPs) containing rLmaC1N and rgp63 recombinant proteins (LPD-rLmaC1N/rgp63) as an effective vaccine. Then, we have investigated the immunogenicity and protective efficacy of LPD-rLmaC1N/rgp63 in experimental animal model of cutaneous leishmaniasis compared to free antigens. Our study showed that mice immunization with LPD-rLmaC1N/rgp63 significantly activates Th1 immune response and enhances IFN-γ production and IgG2a levels. Notably, LPD-rLmaC1N/rgp63 immunization greatly reduced the number of live parasites in the spleen and footpads of immunized mice compared to other vaccinated groups. As well, the thickness of footpad was remarkably smaller in mice immunized with LPD-rLmaC1N/rgp63. In conclusion, our study indicated that LPD-rLmaC1N/rgp63 could be regarded as a promising multiple-antigen candidate vaccine compared to single antigen counterparts to generate protective immunity against leishmaniasis. The potential advantages of this formulation may warrant further investigation.

Cigarette smoking is the leading cause of lung cancer worldwide. In this study, we evaluated the serum autoantibody (AAb) repertoires of non-small cell lung cancer (NSCLC) patients and smokers (SM), leading to the identification of overactivated pathways and hubs involved in the pathogenesis of NSCLC. Surface- and solution-phase biopanning were performed on immunoglobulin G purified from the sera of NSCLC and SM groups. In total, 20 NSCLC- and 12 SM-specific peptides were detected, which were used to generate NSCLC and SM protein datasets. NSCLC- and SM-related proteins were visualized using STRING and Gephi, and their modules were analyzed using Enrichr. By integrating the overrepresented pathways such as pathways in cancer, epithelial growth factor receptor, c-Met, interleukin-4 (IL-4) and IL-6 signaling pathways, along with a set of proteins (e.g. phospholipase D (PLD), IL-4 receptor, IL-17 receptor, laminins, collagens, and mucins) into the PLD pathway and inflammatory cytokines network as the most critical events in both groups, two super networks were made to elucidate new aspects of NSCLC pathogenesis and to determine the influence of cigarette smoking on tumour formation. Taken together, assessment of the AAb repertoires using a systems biology approach can delineate the hidden events involved in various disorders.

Monoclonal antibodies (mAbs) are crucial in pharmaceutical biotechnology. Mammalian cell lines are the most preferred for their production. One of the significant challenging issues of mammalian expression systems is epigenetic gene silencing. Employing epigenetic gene regulation tools can increase the productivity of the mammalian cell lines. Sodium butyrate (NaBut) and valproic acid (VPA) regulate gene expression by inhibiting histone deacetylase. A ubiquitous chromatin-opening element (UCOE) can improve expression by reducing DNA methylation. Here, the separate and combined effects of NaBut and VPA histone deacetylase inhibitors (iHDACs) and UCOE on mAb synthesis were studied. Stable cell lines were generated by non-UCOE (CHO-HL) and UCOE-containing vectors (CHO-UHUL) and cultured in the presence and absence of NaBut or VPA. Expression analysis showed that CHO-UHUL gave a 4-fold greater yield than non-UCOE CHO-HL. Antibody production levels of the CHO-HL and CHO-UHUL cells increased 2-fold and 2.5-fold after NaBut and VPA treatment, respectively. These results indicate that UCOE has more impact on antibody expression than iHDACs. iHDAC treatment exhibited at least a 0.5-fold higher antibody yield in UCOE containing CHO-UHUL cells. Thus utilization of NaBut and VPA (iHDACs) and UCOE resulted in antibody expression improvement, and the combined use of them had a synergistic effect on antibody synthesis. Keywords: Monoclonal antibodies, Chinese hamster ovary, epigenetic gene silencing, chromatin-opening element, sodium butyrate, valproic acid

Background: 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 cell line development and validation in mammalian cells, rational design of CHO cell culture medium or efficient fed-batch strategies are more popular metabolic engineering approaches. Results: 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. Conclusions: Our approach can be used in similar metabolic engineering problems, in order to suggest new ways of increasing production in different cell factories.

Background It is believed that the loading value of anticancer drug conjugated to the monoclonal antibody, called drug-to-antibody ratio (DAR), is the main quality feature of antibody-drug conjugates. Methods In this study, matrix assisted laser desorption/ionization mass spectrometry was used to determine the average molecular weight of trastuzumab and its three conjugated forms. The differences in the measured masses for each conjugate and unconjugated trastuzumab were compared to the expected mass change through the conjugation of one mole of related drug-linker, in order to measure DAR. Results There was a consistency between the loading results of mass spectrometry and the measurements of UV spectrometry in most cases. Conclusion According to our findings, the MALDI-MS method for determining the loading values can be used rapidly and reliably to estimate the covalently bound drugs conjugated to antibodies when ESI-TOF-MS is unavailable.