Kỷ yếu hội nghị Khoa học Môi trường và Công nghệ sinh học năm 2011 161 RECOMBINANT PROTEIN PRODUCTION: EXPRESSION SYSTEMS AND ANIMAL CELL TECHNOLOGY Nguyen Thi Thu Huong (A) Ho Chi Minh City University of Technology (Hutech) TÓM TẮT Protein tái tổ hợp đã và đang đƣợc áp dụng rộng rãi trong lĩnh vực y dƣợc nhằm phòng ngừa và điều trị bệnh. Trong bài báo này, tác giả đã đề cập đến một số dòng tế bào prokaryote và eukaryote hiện đang đƣợc sử dụng nhƣ những hệ thống biểu hiện nhằm thu protein tái tổ hợp. Mỗi dòng tế bào kể trên có ƣu nhƣợc điểm riêng và tùy loại protein tái tổ hợp và mục đích nghiên cứu mà chọn lựa dòng tế bào phù hợp nhất. Bên cạnh đó, còn có các yếu tố khác nhƣ phƣơng pháp chuyển gene vào tế bào chủ, điều kiện và phƣơng pháp nuôi cấy cũng ảnh hƣởng đến việc sàn xuất protein tái tổ hợp ở quy mô lớn. Một số ƣu điểm và nhƣợc điểm/khó khăn có thể gặp phải khi sử dụng tế bào động vật trong quá trình sản xuất protein tái tổ hợp cũng đƣợc đề cập trong báo cáo này. Key words: bioreactor, cell line, culture mode, recombinant protein INTRODUCTION The advance of biotechnology has facilitated the development of recombinant proteins. The number of therapeutic proteins, such as monoclonal antibodies, vaccines has been increased. Several types of prokaryotic and eukaryotic cell lines involve in the production of those pharmaceutical proteins. Each cell line has both benefits and challenges which should be considered before choosing. This literature review will outline some aspects of animal cell technology regarding the production of recombinant proteins. PROTEIN EXPRESSION SYSTEMS Recombinant proteins were developed more than 25 years ago. A large number of them are used as active pharmaceutical ingredients (Gnoth et al., 2008a). Those pharmaceutical proteins have been produced using several expression systems, such as bacteria (E. coli), filamentous fungi and yeast (Saccharyomyces cerevisiae), insect or animal cells (Makrides and Prentice, 2003). Bacterial cells (Escherichia coli) Generally, bacterial cells are the first choice as hosts for expressing foreign proteins (Greene, 2004) due to several reasons. Firstly, it could produce significant amount of recombinant protein quickly (Farrell and Iatrou, 2004). In addition, bacterial cells probably grow at efficient rate in bioreactor. It is also easy to manipulate them genetically. Under the regulation of strong promoters, they could express recombinant proteins in the most efficient and cost-effective manner (Greene, 2004). Therefore, bacteria are the suitable system for high-throughput expression of heterologous proteins producing of recombinant proteins (5 – 50mg) which structural studies often require (Peti and Page, 2007, Davies et al., 2005). However, using bacteria as an expression host reveals a number of disadvantages. For example, bacterial cells often produce the heterologous products in form of inclusion bodies. As a result, downstream processing steps, such as cell disruption, solubilization and refolding, must be undertaken in order to produce recombinant proteins which have clinical efficacy (Gnoth et al., 2008b). In addition, recombinant proteins expressed by bacteria cells are often misfolded, insoluble or inactive (Davies et al., 2005). It is probably because bacteria are incapable of producing eukaryotic post-translational modifications, such as glycosylation, phosphorylation, and amino acid modification (Peti and Page, 2007). Those post-translational activities are crucial for the application of recombinant proteins (Farrell and Iatrou, 2004). Kỷ yếu hội nghị Khoa học Môi trường và Công nghệ sinh học năm 2011 162 Given that, E. coli is the most favoured host cell system for producing foreign proteins if the recombinant proteins do not need posttranslational modifications to obtain efficacy (Gnoth et al., 2008b). Eukaryotic expression system The disadvantages of bacteria system in term of posttranslational modifications could be bridged using the eukaryotic expression systems such as yeast (Saccharyomyces cerevisiae), insect cells and mammalian cells (Greene, 2004).  Saccharyomyces cerevisiae Several benefits have been observed when using Saccharyomyces cerevisiae as an expression host. The first advantage is that it is recognized as a safe organism. It could also process post- translational modifications. Therefore, yeast expression system probably folds, glycosylates and assembles complex recombinant proteins much more efficiently than that of bacteria (Primrose and Twyman, 2006a). In addition, the yeast based expression system may express at high level. Finally, it is easy to scale-up and require inexpensive media in production (Lim and Jin, 2008). In contrast, it is difficult to achieve good excretion when using yeast as expression system (Primrose and Twyman, 2006a). Yeast is also inappropriate when complex glycosylation and posttranslational modifications are required (Shuler and Kargi, 1992).  Baculovirus expression system in insect cells According to Buchs and his colleagues (Buchs et al., 2009), in regard to production of foreign proteins which are then used for structural and functional investigation of therapeutically relevant bio-molecules, baculovirus mediated insect cell expression has become one of the most popular vehicles. It is due to easy scalability and high levels of expression. According to Farrell and Iatrou (2004), a hundred milligrams of recombinant protein per liter of culture medium could be obtained using this expression system. Another advantage is they could perform most of the posttranslational modifications of mammalian cells (McCall et al., 2005). Therefore, using insect cells, a large number of recombinant proteins can be expressed with high functional authenticity (Agathos, 1991). However, there are, at least, several proteins which insect cells could not produce as identical as the native proteins (Shuler and Kargi, 1992). It is probably because of deviations of the posttranslational modification pattern, leading immunogenic (Schmidt, 2004). Regarding gene introduction into large viral genomes, recombinants are generated at a low efficiency, at a frequency of 0.5 – 5% of total virus produced (Primrose and Twyman, 2006b). Other disadvantages include an insufficient expression strength, inefficient processing and impairment of the folding and secretion capacity (Schmidt, 2004). Mammalian cell lines Although the mammalian cell culture system grows slowly, with lower cell density and lower production rate, compared to that of other cell lines (Chun et al., 2001), mammalian cell lines, such as Chinese hamster ovary (CHO), have been used widely as a preferred alternative in production of pharmaceutical proteins (Schmidt, 2004, Davies et al., 2005) due to several reasons. Firstly, they are relatively stable. Secondly, mammalian cell lines process posttranscriptional modification, in particular suitable glycosylation and proper folding of protein produced (Xie et al., 2003). According to Shuler and Kargi (1992), mammalian cells could express protein which is closest to its natural counterpart. Indeed, in some cases, mammalian systems can be the only choice for the preparation of correctly modified proteins (Schmidt, 2004). Finally, it is relatively easy to obtain FDA approval for commercial production using those cell lines. Therefore, utilizing mammalian cell lines has been the best choice for commercial production of many human recombinant proteins, for example blood clotting factors, cytokines, growth factors, immunoglobulins, and thrombolytics (Chun et al., 2001). ANIMAL CELL TECHNOLOGY Animal cell lines  CHO (Chinese hamster ovary) Kỷ yếu hội nghị Khoa học Môi trường và Công nghệ sinh học năm 2011 163 Mammalian cell line represents a preferred alternative in production of pharmaceutical proteins because it is the only cell line that can glycosylate human proteins in the correct manner (Primrose and Twyman, 2006a). Among them, CHO cells, which are often generated by transfection, could be the most widely adopted and utilized for producing recombinant proteins commercially (Bertschinger et al., 2008). Regarding recombinant CHO cell lines, efficiency of protein production is dependent on the fraction of cells carrying transgene in a functional and non- rearranged form. Continuous growth in culture, cell handling, and media manipulations could affect the stability of the inserted sequence (Wurm and Schiffmann, 1999).  Hybridomas Hybridomas are hybrid cell lines, derived from the combination of myeloma, with an infinite lifespan, and B lymphocyte, which could synthesize single antibody (Butler, 2004b). According to Butler (2004b), being grown in suspension in large bioreactors, hybridomas could produce large amount (up to kilogram quantities) of monoclonal antibodies. It is observed that although monoclonal antibodies could be useful in a wide range of applications, such as blood typing, virus detecting, pregnancy testing, due to their high specificity, it is hard to produce antibodies which are not immunogenic to humans. The development of hybridomas has facilitated greatly the production ―humanized‖ antibodies which could be used for treating cancer (Butler, 2004b). Humanized antibodies refer to a chimeric antibody that the variable regions derived from mouse are linked to human constant regions. The hybrid antibodies have been applied as human therapeutic agents. Using this particular antibody could reduce an undesirable immune reaction which often occurs when using monoclonal antibodies derived from mouse (Butler, 2004b). Gene transfer Introduction of foreign DNA into mammalian or insect cells could be implemented using several ways. In biological mechanism, target cells are infected with a biological delivery vector, such as a virus (transduction) or bacterium (bactofection), carrying the exogenous genetic material.  Transduction. Interested gene could be added to the intact genome of virus or could replace one or more viral genes. Utilizing naturally infectional and transfectional ability of virus, the transgene is delivered into animal cells as part of a recombinant viral genome (Primrose and Twyman, 2006b).  Bactofection. This method involves in the use of bacteria which could invade animal cells. Using plasmid carried by bacterium, the transgene is then transferred into animal cells (Primrose and Twyman, 2006b). In regard to non-biological mechanism, there are two ways to insert foreign DNA into animal cells  Direct transfer by physical transfection. Using physical transfection methods, such as microinjection, particle bombardment, ultrasound, and electroporaion, naked DNA is transferred into the animal cells directly (Primrose and Twyman, 2006b).  Chemical-mediated transfection. DNA could be uptaken into cells as a synthetic complex. That is DNA, as a complex, has positive charge, so it could interact with the negatively charged cell membrane. Consequently, it promotes uptake by endocytosis. Alternatively, DNA can play a role as a lipophilic complex fusing with membrane. At a result, it deposits the transgene into the cytoplasm directly (Primrose and Twyman, 2006b). Bioreactor  Stirred tank The number of reactor types has been increased. However, because of the simplicity and the ease of documentation, in comparison to other bioreactors, the conventional stirred tank still represents as the preferred choice in industrial scale (Persson and Emborg, 1992). Because mammalian cell does not have cell wall which could help to resistant to strong shear force resulted by high agitation (Xing et al., 2009), animal cells are shear sensitive and increasing Kỷ yếu hội nghị Khoa học Môi trường và Công nghệ sinh học năm 2011 164 shear forces and longer shear times could cause cellular damage (Pol et al., 1990). Therefore, searing and bubble damage could raise the concern in large-scale systems. However, this issue could be solved by adding surfactants, such as Pluronic-F68.  Wave bags The Wave Bioreactor is a recent development in cell cultivation technology. It includes a sterile and disposable cell bag placed on a rocking thermo-platform. In production, the cell bag is filled with media partially. The advantages of using the wave bioreactor make the system highly attractive over traditional systems for animal cell culture such as shake flasks and stirred tanks (Mikola et al., 2007).  After using, the material will be discarded, leading the reduction of the need for cleaning or validation steps, thereby significantly reducing costs in cGMP operations (Mikola et al., 2007, Hanson et al., 2009).  It can be installed and used rapidly for process development and clinical manufacturing, hence reducing the time to market for biological products (Mikola et al., 2007).  The rocking motion could create waves imparting mixing and promoting oxygen transfer (Mikola et al., 2007). Consequently, cells are not exposed to large variations in shear forces and hence they could grow in a more stable physical environment (Slivac et al., 2006)  The production process could be monitored (Mikola et al., 2007).  The system is widely used in cell culture due to ease of use (Mikola et al., 2007). Culture modes in large scale  Batch culture After being introduced into bioreactor, the cells are inoculated and the culture is left for several days. The production process finishes when the final density is reached. In this closed system, apart from tiny amount of samples for analysis, nothing is added or removed during the culture (Butler, 2004c). Due to its simplicity, this mode is used widely in production of FDA- approved therapeutic proteins (Xie et al., 2003).  Fed batch culture Fed-batch process, which has been used widely for large scale production of therapeutic proteins cells (Ye et al., 2009), is a mode that a feed stream which contains substrate and nutrient is supplied to bioreactor during the period of batch fermentation (Meszaros and Bales, 1992) in order for the cells to grow effectively and increase cell life, thereby considerably improving productivity (Ye et al., 2009). After the production process, the culture is discarded partially or completely, and the operation is repeated (Meszaros and Bales, 1992). Controlling the substrate concentration helps to overcome several effects, for instance, substrate inhibition, catabolite repression, product inhibition, glucose effect, and autotrophic mutation (Meszaros and Bales, 1992).  Perfusion culture In this mode, cells in the harvest stream are maintained or recycled back to the bioreactor, while product is harvested continuously from the vessel. Fresh culture medium is also added to maintain a constant culture volume in a continuous culture (Xie et al., 2003). Serum-free media In order to grow animal cell line in vitro, it is necessary to use a mixture of nutrients, named cell culture media. Generally, they include glucose, amino acids, vitamin and salt (Xie et al., 2003). Serum is one of important components in cell culture media providing growth and adhesion factors, low molecular-weight nutrients, hormones, and growth factors (Park et al., 2006). However, it probably contains many undefined components with a variety of specific and non-specific effects on cells (Pol et al., 1990) resulting in obstacles to the production of medically useful proteins in animal cell culture systems (Park et al., 2006). Hence, removing animal sera and other animal derived components from culture media has been increasing (Xie et al., 2003). Consequently, serum-free media or serum-supplemented media have been developed (Park et al., 2006). Serum-free media are often more complicated and include pure recombinant growth factors, such as insulin-like growth factor (IGF) to maintain the highest quality standards. These growth Kỷ yếu hội nghị Khoa học Môi trường và Công nghệ sinh học năm 2011 165 factors play an important role in mammalian growth and development. Lack of them could reduce the cell growth-promoting activity of a culture by as much as 90% (Park et al., 2006). The advantages and disadvantages/challenges Animal cell culture system has been used widely for production of recombinant proteins due to following reasons:  They could process authentic post-translational modifications of recombinant proteins, which bacteria do not carry out (Primrose and Twyman, 2006b). Consequently, it could reduce the risk of formation of structurally altered compounds with immunogenic properties (Schmidt, 2004).  Mammalian cells, such as CHO and BHK, are recognized as safe in regard to infectious and pathogenic agents. Hence, recombinant proteins expressed by those cell lines could be likely approved by regulatory bodies (Schmidt, 2004). Along with the advantages there are several disadvantages and challenges which could suffer from when using animal cell in the production of recombinant proteins.  Due to low yield and complicated and costly cultivation, therefore animal cells are probably used when they are the only choice for the preparation of correctly modified proteins (Schmidt, 2004).  Mammalian cells could respond to various changes such as nutrient deprivation, oxygen limitations, toxin accumulations, osmolarity increasing (Jr. et al., 2007).  They require specialized media and sufficient oxygen supply (Sandig et al., 2005).  Animal cells have low cell density and grow at slow kinetics (Sandig et al., 2005).  They are also high sensitive mechanical stress (Sandig et al., 2005).  According to Butler (2004a), contamination is one of reasons resulting the failure in operation of cell cultures. Growing at slow rate could make culture of animal cells be vulnerable to microbial contaminants (Butler, 2004a). Another factor resulting in the high rate of contamination in the culture media is the inclusion of animal sera and other animal-derived raw materials (Xie et al., 2003). Some components in culture medium could be heat-sterilized to prevent contamination, while due to the natural features, several constituents should be sterilized by filtration or irradiation (Xie et al., 2003).  Their information of genome has not been investigated completely (Park et al., 2006). REFERENCES Agathos, S. N. (1991) Production scale insect cell culture. Biotech. Adv. , 9, 51 - 68. Bertschinger, M., Schertenleib, A., Cevey, J., Hacker, D. & Wurm, F. (2008) The Kinetics of Polyethylenimine-Mediated Transfection in Suspension Cultures of Chinese Hamster Ovary Cells. Molecular Biotechnology, 40, 136-143. Buchs, M., Kim, E., Pouliquen, Y., Sachs, M., Geisse, S., Mahnke, M. & Hunt, I. (2009) High- Throughput Insect Cell Protein Expression Applications. High Throughput Protein Expression and Purification. 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Ye, J., Kober, V., Tellers, M., Naji, Z., Salmon, P. & Markusen, J. F. (2009) High-level protein expression in scalable CHO transient transfection. Biotechnology and Bioengineering, 103, 542- 551. . 2011 161 RECOMBINANT PROTEIN PRODUCTION: EXPRESSION SYSTEMS AND ANIMAL CELL TECHNOLOGY Nguyen Thi Thu Huong (A) Ho Chi Minh City University of Technology (Hutech) TÓM TẮT Protein tái. literature review will outline some aspects of animal cell technology regarding the production of recombinant proteins. PROTEIN EXPRESSION SYSTEMS Recombinant proteins were developed more than 25. disadvantages and challenges which could suffer from when using animal cell in the production of recombinant proteins.  Due to low yield and complicated and costly cultivation, therefore animal cells