RESUMO
Protein L affinity chromatography is a useful method for the purification of antibody fragments containing kappa light chains. In affinity chromatography, increasing the binding affinity leads to increased product purity, recovery, and dynamic binding capacity (DBC). In this study, molecular docking and molecular dynamics simulation techniques were used to design the engineered Protein L with higher affinity to the kappa light chain. Each engineered ligand was produced as a recombinant protein and coupled to a solid matrix. The purity, recovery, and DBC of the engineered resins were evaluated and then compared to those of a commercially available resin. The results showed important parameters for engineering more efficient Protein L ligands for affinity chromatography.
Assuntos
Fragmentos de Imunoglobulinas , Cromatografia de Afinidade , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
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.
Assuntos
Escherichia coli/genética , Proteínas Recombinantes de Fusão , Fator de Necrose Tumoral alfa , Animais , Linhagem Celular , Cromatografia de Afinidade , Meios de Cultura/metabolismo , Humanos , Camundongos , Redobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/isolamento & purificação , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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.
Assuntos
Anticorpos Monoclonais/biossíntese , Reatores Biológicos , Técnicas de Cultura de Células , Animais , Anticorpos Monoclonais/genética , Células CHO , Cricetulus , Meios de Cultura , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
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.
Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Listeria monocytogenes/imunologia , Ativação Linfocitária/imunologia , Fator Tu de Elongação de Peptídeos/imunologia , Proteínas de Bactérias/imunologia , Linhagem Celular , Células Dendríticas/citologia , Citometria de Fluxo , Humanos , Imunoterapia/métodos , Teste de Cultura Mista de Linfócitos , Receptores de Reconhecimento de Padrão/imunologiaRESUMO
Because of the lack of an accurate and sensitive tool to evaluate the parasitemia level, treatment or prevention of leishmaniasis remains an important challenge worldwide. To monitor and track leishmanial infection by two parameters in real time, we generated stably transgenic Leishmania that express a bi-reporter protein as fused EGFP and firefly luciferase. Using two reporter genes (egfp-luc) simultaneously increases the experimental sensitivity for detection/diagnosis, and in vitro quantification of parasites as well as real-time infection in mice. Through different specific tools, EGFP and LUC signals from the parasite were detectable and measurable within a mammalian host and promastigotes. Here, the LUC protein provided a higher level of sensitivity than did EGFP, so that infection was detectable at an earlier stage of the disease in the footpad (injection site) and lymph nodes by bioluminescence. These results depicted that: (1) both quantitative reporter genes, EGFP and LUC, could be simultaneously used to detect parasitemia in vitro and in vivo and (2) sensitivity of firefly luciferase was 10-fold higher than that of EGFP in promastigotes.
Assuntos
Genes Reporter , Proteínas de Fluorescência Verde/genética , Leishmania major/genética , Leishmaniose Cutânea/parasitologia , Luciferases de Vaga-Lume/genética , Anfotericina B/farmacologia , Anfotericina B/uso terapêutico , Animais , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Feminino , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Leishmania major/efeitos dos fármacos , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/tratamento farmacológico , Luciferases de Vaga-Lume/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Organismos Geneticamente Modificados , Fatores de Tempo , TransfecçãoRESUMO
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.
Assuntos
Proteínas do Citoesqueleto/análise , Expressão Gênica , Interações Hospedeiro-Patógeno , Neurônios/química , Neurônios/virologia , Vírus da Raiva/crescimento & desenvolvimento , Animais , Linhagem Celular , Eletroforese em Gel Bidimensional , Espectrometria de Massas , Camundongos , ProteômicaRESUMO
Protein L is a multidomain protein from Peptostreptococcus magnus with binding affinity to kappa light chain of human immunoglobulin (Ig) which is used for the purification of antibody fragments by affinity chromatography. The advances in protein engineering and computational biology approaches lead to the development of engineered affinity ligands with improved properties including binding affinity. In this study, molecular dynamics simulations (MDs) and Osprey software were used to design single B domains of the Protein L with higher affinity to antibody fragments. The modified B domains were then polymerized to ligand with six B domains by homology modeling methods. The results showed that single B domain mutants of MB1 (Thr865Trp) and MB2 (Thr847Met-Thr865Trp) had higher binding affinity to Fab compared to the wild single B domain. Also, MDs and molecular docking results showed that the polymerized Proteins L including the wild and mutated six B domains (6B0, 6B1, and 6B2) were stable during MDs and the two mutants of 6B1 and 6B2 showed higher binding affinity to Fab relative to the wild type.Communicated by Ramaswamy H. Sarma.
RESUMO
Background: Post-translational modifications in bioprocessing and storage of recombinant mAbs are the main sources of charge variants. While the profile of these kinds of variants is considered an important attribute for the therapeutic mAbs, there is controversy about their direct role in safety and efficacy. In this study, the physicochemical and pharmacokinetic (PK) properties of the separated charge variants belonging to a trastuzumab potential biosimilar, were examined. Methods: The acidic peaks, basic peaks, and main variants of trastuzumab were separated and enriched by semi-preparative weak cation exchange. A panel of analytical techniques was utilized to characterize the physicochemical properties of these variants. The binding affinity to HER2 and FcγRs and the PK parameters were evaluated for each variant. Results: Based on the results, the charge variants of the proposed biosimilar had no significant influence on the examined efficacy and PK parameters. Conclusion: During the development and production of biosimilar monoclonal antibodies, evaluating the effect of their charge variants on efficacy and PK parameters is needed.
Assuntos
Medicamentos Biossimilares , Trastuzumab/química , Medicamentos Biossimilares/química , Medicamentos Biossimilares/farmacocinética , Anticorpos MonoclonaisRESUMO
Human epidermal growth factor receptor 2 (HER2) overexpression has been demonstrated in a variety of cancers. Targeted therapy with anti-HER2 monoclonal antibodies (mAbs) has been approved as a therapeutic modality. Despite the efficacy of mAbs in tumor treatment, many patients do not benefit from this therapeutic platform. Fragment crystallizable (Fc) engineering is a common approach to improve the efficacy of therapeutic mAbs. Five Fc-engineered mAbs have so far been approved by FDA. We have recently developed an anti-HER2 bispecific mAb, BiHT, constructed from variable domains of trastuzumab, and our novel humanized anti-HER2 mAb, hersintuzumab. BiHT displayed promising antitumor activity as potently as the combination of the parental mAbs. Here, we aimed to modify the Fc of BiHT to improve its therapeutic efficacy. The Fc-engineered BiHT (MBiHT) bound to recombinant HER2 and its subdomains with an affinity similar to BiHT. It also recognized native HER2 on different cell lines, inhibited their proliferation, downregulated HER2 expression, and suppressed downstream signaling pathways similar to BiHT. Compared with BiHT, MBiHT displayed enhanced antibody-dependent cellular cytotoxicity activity against various tumor cell lines. It also inhibited the growth of ovarian xenograft tumors in nude mice more potently than BiHT. Our findings suggest that MBiHT could be a potent therapeutic candidate for the treatment of HER2-overexpressing cancer types.
Assuntos
Anticorpos Biespecíficos , Anticorpos Monoclonais Humanizados , Camundongos , Animais , Humanos , Camundongos Nus , Trastuzumab , Anticorpos Monoclonais/metabolismo , Receptor ErbB-2 , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Background: Background: Downstream processing of therapeutic recombinant proteins expressed as the inclusion bodies (IBs) in E. coli is quite challenging. This study aimed to use the quality by design approach for developing the multi-step downstream process of a structurally complex therapeutic Fc-Peptide fusion protein, romiplostim. Methods: Methods: For development of a successful downstream process, risk analysis and experimental designs were used to characterize the most critical quality attributes (CQAs) and effects of process parameters on these quality attributes. Results: Results: The solubilization of IBs was optimized by design of experiment on three parameters with a focus on solubility yield, which resulted in >75% increase of the target protein solubilization. The pH of sample was identified as CQA in anion exchange chromatography that might have an impact on achieving >85% host cell proteins removal and >90% host cell DNA reduction. In the refolding step, process parameters were screened. Cystine/cysteine ratio, pH, and incubation time identified as CPPs were further optimized using Box-Behnken analysis, which >85% of the target protein was refolded. The design space for further purification step by HIC was mapped with a focus on high molecular weight impurities. After polishing by gel filtration, the final product's biological activity showed no statistically significant differences among the groups received romiplostim and Nplate®, as the reference product. Conclusions: Conclusion: This research presents a precise and exhaustive model for mapping the design space in order to describe and anticipate the link between the yield and quality of romiplostim and its downstream process parameters.
Assuntos
Escherichia coli , Corpos de Inclusão , Proteínas Recombinantes de Fusão , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão/biossínteseRESUMO
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.
Assuntos
Proteínas de Fluorescência Verde/biossíntese , Leishmania/metabolismo , Leishmaniose/parasitologia , Macrófagos/parasitologia , Animais , Western Blotting , Linhagem Celular , Separação Celular , Células Cultivadas , Clonagem Molecular , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Expressão Gênica , Genes de Protozoários , Genes Reporter , Proteínas de Fluorescência Verde/genética , Processamento de Imagem Assistida por Computador , Leishmania/genética , Leishmania infantum/genética , Leishmania infantum/metabolismo , Leishmania major/genética , Leishmania major/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Organismos Geneticamente Modificados , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransgenesRESUMO
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.
Assuntos
Antagonistas Adrenérgicos beta/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Carvedilol/administração & dosagem , Trastuzumab/efeitos adversos , Disfunção Ventricular Esquerda/prevenção & controle , Administração Oral , Animais , Cardiotoxicidade/diagnóstico , Cardiotoxicidade/etiologia , Cardiotoxicidade/prevenção & controle , Modelos Animais de Doenças , Ecocardiografia , Feminino , Humanos , Injeções Intraperitoneais , Pessoa de Meia-Idade , Ratos , Volume Sistólico/efeitos dos fármacos , Trastuzumab/administração & dosagem , Disfunção Ventricular Esquerda/induzido quimicamente , Disfunção Ventricular Esquerda/diagnósticoRESUMO
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.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Antineoplásicos Imunológicos/toxicidade , Carvedilol/farmacologia , Mioblastos Cardíacos/efeitos dos fármacos , Proteoma/metabolismo , Trastuzumab/toxicidade , Antagonistas Adrenérgicos beta/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Cardiomiopatias/prevenção & controle , Carvedilol/uso terapêutico , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Simulação por Computador , Regulação para Baixo , Feminino , Humanos , Mioblastos Cardíacos/metabolismo , Mioblastos Cardíacos/patologia , Proteômica , Receptor ErbB-2/metabolismo , Trastuzumab/uso terapêuticoRESUMO
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.
Assuntos
Certolizumab Pegol , Polietilenoglicóis , Animais , Artrite Reumatoide/tratamento farmacológico , Linhagem Celular , Certolizumab Pegol/química , Certolizumab Pegol/farmacocinética , Certolizumab Pegol/farmacologia , Doença de Crohn/tratamento farmacológico , Feminino , Meia-Vida , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Polietilenoglicóis/farmacologiaRESUMO
The crucial role of VEGF receptor 2 (VEGFR2) signaling in the angiogenesis and metastasis of solid tumors has prompted the development of inhibitors with minimal bystander effects. Recently, Adnectin C has attracted attention for cancer treatment. To overcome the problematic properties of Adnectin, a novel form of Adnectin C has been designed by its fusion to a biodegradable polymeric peptide containing Pro/Ala/Ser (PAS) repetitive residues. E. coli-expressed recombinant fused and unfused proteins were compared in terms of bioactivity, physicochemical, and pharmacokinetic properties using standard methods. Dynamic light scattering (DLS) analysis of PASylated adnectin C revealed an approximate 2-fold increase in particle size with a slight change in the net charge. Additionally, fusion of the PAS sequence improved its stability against the growth of thermo-induced aggregated forms. The high receptor-binding and improved binding kinetic parameters of PASylated Adnectin C was confirmed by ELISA and surface plasmon resonance assays, respectively. Pharmacokinetic studies showed a noticeable increase in the terminal half-life of Adnectin C-PAS#1(200) by a factor of 4.57 after single dose by intravenous injection into female BALB/c mice. The results suggest that PASylation could offer a superior delivery strategy for developing Adnectin-derived drugs with improved patient compliance.
Assuntos
Fibronectinas/farmacologia , Fibronectinas/farmacocinética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Alanina , Animais , Escherichia coli , Feminino , Fibronectinas/isolamento & purificação , Fibronectinas/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/farmacocinética , Fragmentos de Peptídeos/farmacologia , Prolina , Domínios Proteicos/fisiologia , Engenharia de Proteínas/métodos , Serina , Ressonância de Plasmônio de Superfície/métodos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.
Assuntos
Aminoácidos/metabolismo , Anticorpos Monoclonais/biossíntese , Sobrevivência Celular/fisiologia , Proteômica/métodos , Aminoácidos/farmacologia , Animais , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , CamundongosRESUMO
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.
RESUMO
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.
Assuntos
Aminoácidos/farmacologia , Bevacizumab/biossíntese , Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Engenharia Genética , Análise de Variância , Animais , Bevacizumab/genética , Células CHO , Cricetinae , Cricetulus , Imunoglobulina G/metabolismo , Polissacarídeos/metabolismoRESUMO
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.
RESUMO
BACKGROUND: Novel vaccination approaches are needed to prevent leishmaniasis. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The nonpathogenic to humans lizard protozoan parasite, Leishmania (L) tarentolae, has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with a salivary protein has been shown to protect mice against cutaneous leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: Here, we tested the efficacy of a novel combination of established protective parasite antigens expressed by L. tarentolae together with a sand fly salivary antigen as a vaccine strategy against L. major infection. The immunogenicity and protective efficacy of different DNA/Live and Live/Live prime-boost vaccination modalities with live recombinant L. tarentolae stably expressing cysteine proteinases (type I and II, CPA/CPB) and PpSP15, an immunogenic salivary protein from Phlebotomus papatasi, a natural vector of L. major, were tested both in susceptible BALB/c and resistant C57BL/6 mice. Both humoral and cellular immune responses were assessed before challenge and at 3 and 10 weeks after Leishmania infection. In both strains of mice, the strongest protective effect was observed when priming with PpSP15 DNA and boosting with PpSP15 DNA and live recombinant L. tarentolae stably expressing cysteine proteinase genes. CONCLUSION/SIGNIFICANCE: The present study is the first to use a combination of recombinant L. tarentolae with a sand fly salivary antigen (PpSP15) and represents a novel promising vaccination approach against leishmaniasis.