ABSTRACT
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
ABSTRACT
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
ABSTRACT
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
ABSTRACT
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 IgG[4] in Sp2.0 cells. For this purpose, the recombinant Sp2.0 cells producing an anti CD33 IgG[4], were subjected to different concentrations of cysteamine. At concentrations of 2, 4 and 5 mM cysteamine, the secreted levels of IgG[4] did not change significantly. However, in concentration of 7 mM cysteamine, a significant decrease was observed in IgG[4] 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 IgG[4] in this type of recombinant cells
Subject(s)
Cell Growth Processes/drug effects , Cell Survival/drug effects , Immunoglobulin GABSTRACT
The study of physiological changes in recombinant cell lines provides useful information to improve production performance. In this study, we investigate the effects of an anti-CD33 chimeric IgG4 expression on Sp2.0 cell growth. Variable region genes of light and heavy chains of monoclonal antibody produced by M195 were cloned in pFUSE-CLIg-hk and pFUSE-CHIg-hG4 expression vectors, respectively. Transfection of recombinant plasmids into Sp2.0 cell lines was performed using lipofectamine in two steps. Positive transformant cells were isolated and subjected to PCR, RT-PCR and Western blot analysis to confirm the integration of gene cassettes and the expression of recombinant IgG4. To assess the growth parameters, recombinant and parent Sp2.0 cell lines were seeded at a density of 1×10[5] cells/ml in duplicate into 12-well plates. For nine days, culture plates were sampled daily and viable cell count and viability determined. The results of PCR, RT-PCR and Western blot analyses confirmed the generation of stable producer cell lines. In recombinant cells, the maximum cell density decreased by 46%. However, it was observed that IgG4 expression had no effect on cell viability of these transfectants. Our results showed that the expression of recombinant IgG4 can change growth parameters in Sp2.0 cell lines that express the pFUSE-CHIg-hG4-pFUSE-CLIg-hk construct
ABSTRACT
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. 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. 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. 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
ABSTRACT
Development of high producing mammalian cell lines is a major bottleneck in manufacturing of recombinant therapeutic proteins. This study examines the effect of using the matrix attachment region from the human interferon beta gene in combination with promoter activation strategy with E1A 13S protein on human tissue plasminogen activator [t-PA] expression in Chinese hamster ovary [CHO] cells. The matrix attachment region was cloned in 3' and 5' flanking sides of the t-PA expression cassette in pTPA vector to generate pMTPA. After transfection of the cells with pTPA and pMTPA vectors, stable cell pools were developed and the t-PA expression level determined for each stable cell line. In the next step, E1A 13S expression plasmid was transfected to stable cell pools and t-PA titers were measured after 72 hours. Integration of pTPA and pMTPA vectors in the CHO genome was confirmed by PCR analysis on genomic DNA of stable cell pools. Analysis of the t-PA expression level showed a three-fold enhancement in pMTPA transfected cells compared to pTPAcontaining cells. t-PA expression was further enhanced up to 1771 U/ml by transient expression of E1A 13S in pMTPA stable cell pools. These results have shown that incorporation of matrix attachment region in an expression vector in combination with promoter activation can effectively enhance recombinant protein expression levels in CHO cells.