A Review of Metabolic and Cell Engineering of Mammalian Cells

Name: Paul S McCarthy Student No: 10274731

Metabolic and Cell Engineering of Mammalian Cells

Table of Contents
1. 2. 2.1. 2.2. 2.2.1. 2.2.2. 2.2.3. 2.2.4. 2.2.5. 2.2.5.1. 2.2.5.2. 2.2.5.3. 2.2.6. 3. 3.1. 3.2. 3.3. Introduction ................................ ................................ ................................ ........................... 3 Cell Engineering ................................ ................................ ................................ ..................... 3 Cell Lines ................................ ................................ ................................ ............................ 4 Expression Systems ................................ ................................ ................................ ............ 5 Expression Vectors ................................ ................................ ................................ ......... 5 Transfection ................................ ................................ ................................ ................... 5 Transfection Methods................................ ................................ ................................ ..... 7 Markers ................................ ................................ ................................ .......................... 8 DNA delivery systems ................................ ................................ ................................ ..... 8 Calcium-phosphate Precipitation ................................ ................................ ................ 9 Electroporation................................. ................................ ................................ .......... 9 Lipofection and polyfection................................. ................................ ........................ 9 Cell Selection ................................ ................................ ................................ .................. 9

Metabolic Engineering ................................ ................................ ................................ ......... 10 Apoptosis ................................ ................................ ................................ ......................... 10 Engineering cells for hypothermic growth ................................ ................................ ........ 11 Glycosylation ................................ ................................ ................................ .................... 12

Bibliography ................................ ................................ ................................ ................................ .... 12

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1. Introduction
The advent of recombinant DNA technology enabled heterologous gene expression in living cells and thereby large-scale production of protein therapeutics. Many protein pharmaceuticals are produced using bacterial and yeast cells because of their rapid growth and high expression levels. However, many of the most promising protein therapeutics require post-translational modifications such as glycosylation for full therapeutic efficacy. Therefore, mammalian cells are rapidly becoming the standard for industrial production of pharmaceutical proteins. Chinese hamster ovary (CHO) cells have emerged as the most widely used industrial production cell line since it is:
   

Well characterized; Known for the stability of chromosomal transgene integrations; Easy-to-cultivate at large scale; and Simple to adapt for suspension growth in serum- and protein-free media.

The expanding list of therapeutic proteins produced by mammalian cell culture processes includes blood clotting factors, monoclonal antibodies and thrombolytic. As of the beginning of the new millennium more than 100 biological were either in phase III clinical trials or awaiting FDA approval. An annual approval rate of up to 10 therapeutic monoclonal antibodies is expected in the years to come]. Large-scale production of monoclonal antibodies is particularly challenging because the average dosing regimens are typically up to an order of magnitude higher compared to other protein therapeutics. Considering these developments in the biotechnology industry as we as the high ll cost/low yield characteristics of mammalian cell culture processes compared to bacterial or yeast fermentations, there is increasing demand for optimization of various production parameters including cell specific productivity and product quality (Kaufmann, et al., 2003). Approach to increase production of monoclonal antibody throu optimizing growth conditions, gh media compositions and process control have all imporved the yields, but the use of recombinant DNA technology enables heterlogous gene expression in living cells and thereby large scale production of protein therapeutics (Kaufmann, et al., 2003). There are a number of hurdles that must be overcome, such as extensive screening of clones, regulation of gene expression, silencing of genes, loss of gene copies and location of transgenic integration site, all of these elements can create a unstable cell or a variable expression in the cell (Al-Rubeai, 2010).

2. Cell Engineering
Cell Engineering aims at changing the gene expression program of specific host cell line for instance by molecule techniques (Imhof, et al., 2009), thereby improving the production of therapeutic proteins. The development of improved production of monoclonal antibodies and other Paul S McCarthy Page 3 of 13 10274731

Metabolic and Cell Engineering of Mammalian Cells recombinant proteins is often associated with progress in cell culture technology and particularly in mammalian cell culture. The production of monoclonal antibodies in mammalian cells consists of a long process that involves steps of transfection of the gene of interest into the cells, selection of clones, adaptation to different culture conditions (usually suspension and se rum-free medium), culture in bioreactors and scale-up to industrial level. Cell engineering aim is to improve production f monoclonal anti body production by improving cell line types, cell size, cell cycle, product characterictics, vector / promoter of transfection, methods of transfection and clone selection, post transcription regulation and growth medium. It is these factors that will allow us to improve production. Mammalian cells are the primary host for production of therapeutic proteins such as monoclonal antibodies, but a variety of perkaryotic and eukaryotic cells / systems have been used. The advantage of mammalian cells it that they have the ability to perform human like post translational modifications. The product of monoclonal antibodies in these cells begins with the selection of a suitable cell line and type. The chosen cell must have the following characteristics  Support high level product expression over long periods of time, maintaining high viable cell density and genetic stability  Scalable  Appropriate abilities for post translational processing  Allow appropriate characterization for human safety (Ozturk, et al., 2006)

2.1.

Cell Lines

The type of cell lines to use can be determined by the amount of product you may wish to produced, for example Mosser et al states that for small scale quantities of monoclonal anti body for the biochemical and biophysical analysis can be produced by African green monkey kidney (COS) cell line (Mosser, et al., 1994). COS can not be used for large scale production due to the fact that they louse production ability over time. The most commonly used cells for large scale production are Chinese Hamster Ovary cells (CHO). There suitability for large scale and stable production of monoclonal anti body is related to their advantages of safety for use in humans especially the resemblance between glycan structure of their product with natural human monoclonal antibodies, their ease f transfection, presence of gene amplification systems, ease of adaptation to growth in either suspension and serum free medium and the ability to grow too high cell densities (Trill, et al., 1995) (Wurm, 2004). Mycloma cells like SP 2/0, YB 2/0 and NSO are used for large scale production; they are similar to CHO cells. They are capable to grow to high cell densities in either suspension or serum free medium and most importantly can be readily transfected. NSO cell line is used in the production of Remicade (Yang, et al., 2007). Hybridoma cell are used as well, but they are extremely sensitive to their environmental changes and form toxic compounds during cell growth. Also hybridomea cells are very sensitive to shear and bubble damage in reactors. Other cell lines that can be used are Baby Hamster Kidney cells (BHK), Human Embryonic Kidney Cells and Human Retina-derived PER-C6 cells which have shown some promise. They been shown to have high yield capabilities of recombinant products such as monoclonal anti body, they are easily adaptable to different growth conditions and doesn t require gene amplification or adding non Paul S McCarthy Page 4 of 13 10274731

Metabolic and Cell Engineering of Mammalian Cells human glycan structure to the protein. To achieve a high yield it is more important to have a combination of host cell, expression vector, transfection and selection strategy rather than relying solely on host characteristics.

2.2.

Expression Systems

2.2.1. Expression Vectors

Expression Vectors are required for the expression of heterologous protein in mammalian cells in order to transfer the product gene into the cell. There are core elements expression vectors should have,
 Expression levels independent from the site of integration in the genome  Expression levels correlated with the number of integrated copies  Maintain expression efficiency over time (Costa, et al., 2010)

Due to the specific requirements, mammalian vectors are usually plasmids. Plasmids are circular DNA molecules that exist in bacterial cells apart from their main chromosome and possess at least one replication origin. This allows plasmids to replicate within cells, independently from bacterial or eukaryotic chromosomes.

2.2.2. Transfection
The ability to express genes in mammalian cells requires a cassette that consists of two types of elements: promoter/ enhancer elements that drive mRNA transcription and sequences that help to stabilize or enhance translation of the primary transcript. Promoters/enhancers: promoters are elements that drive the expression of the recombinant gene, promoting and accurately positioning the beginning of transcription, while enhancers are elements that augment transcription. For the expression of recombinant proteins in mammalian cells, a strong viral promoter / enhancer or a cellular promoter/enhancer combination known to be particularly active to the host cell are commonly used (Ozdemir, 1998). The two most common enhancers are sourced from the Simian virus 40 (SV40) and Cytomegalovirous (CMV), others include Visna virus and CHO elongation factor-alpha (EF1- ). The aim we are trying to achieve is to gain a stabilised and enhanced translation of t e primary h transcript. One such method is the use of polyadenylation signals which are taken from SV40 and bovine growth hormone gene, which are common in mammalian expression systems. It is thought that they prolong the half life of mRNA in cytoplasm and to carry out efficient translation. Another method is the Kozak sequence, which is the optimum consensus sequence for surrounding the initiator codon of mammalian genes and is used to improve the translation initiation of mRNA of the gene of interest (Costa, et al., 2010) Paul S McCarthy Page 5 of 13 10274731

Metabolic and Cell Engineering of Mammalian Cells The gene of interest is isolated as a cDNA without introns, but by including at least one intron between the promoter and the cDNA coding sequence, we can achieve an increase of efficiency in the cytoplasmatic transport and higher steady state levels of cytoplasmatic mRNA, that would be available for translation. When the mammalian expression vector is used for generation of a stable producing cell line, an extra sequence which includes the encoding for a selectable marker gene, is used. This selection or marker gene can be present on the same vector as the recombinant gene or in separate vectors, and can be driven from a weak promoter, in order to increase the possibility to obtain high level of producer cells (Costa, et al., 2010). But this usually reduces the efficiency of stable transfection, with decreased numbers of surviving cells, but those that survive selection have the ability to produce higher quantities of the recombinant product. Furthermore, to improve the efficiency of stable transfection, plasmids can be linearized before this process using restriction enzymes. The plasmid DNA molecules which contain the gene of interest will be randomly integrated into the host genome. This method of random integration is simple and straight forward, but lacks reproducibility which is not our aim. Costa et al states that the site of integration mostly influences the transcription rate of the recombinant gene, a phenomenon known as the position effect. Depending on the surrounding chromatin at the integration site, expression of the product can be high, low or even null, the expression of the recombinant gene tends to be inactivated (silenced) over time and this consequently makes the selection of suitable high-producing clones more tiresome and time-consuming (Costa, et al., 2010). To overcome the position effects, different strategies have been developed, such strategies include the use of anti-repressor elements on either side of the vectors, this strategy is called Expression Augmenting Sequence Elements EASE (Kwaks, et al., 2003). The integration of vectors specifically into chromosomal loci with open chromatin or through the use of nuclear regulatory factors such as Matrix-attachment regions (MARS) or Scaffoldattachment regions (SARS)) (Costa, et al., 2010). There are situations where a stable and continuous production is not desired and a regulated or induced gene expression is needed (Ozturk, et al., 2006). For this purpose, inducible mammalian expression systems have been developed. Initially, they relied on inducers with pleiotropic effects on host cells, such as heat shock induction of expression with its promoter, heavy metal induction of the metallothionein promoter or glucocorticoid induction of steroid responsive promoters. However, due to the broad range of effects that they produced and the high basal expression, new inducible systems have been developed, including the Lac/IPTG, the tetracycline (tet), the streptogramin and the ecdysone systems (Costa, et al., 2010). All these systems use a regulatory plasmid for the expression of a repressor or trans-activator and an expression plasmid containing the regulated promoter linked to the product gene (Ozturk, et al., 2006). The activity of the repressor or activator is controlled by the addition of a specific compound to the culture medium such as IPTG thereby controlling the promoter and the expression of the gene of interest / product. (Bi, et al., 2004). Apart from the problems in the design of expression vectors, that are common for all recombinant proteins, monoclonal antibodies have additional issues that cause concern such as their structure of heavy (HC) and light (LC) chain subunits. These subunits interaction can influence the kinetics of monoclonal antibody construction in the cell which can results in the need to express these subunits at optimal stoichiometric ratios (Costa, et al., 2010). This has led to the development of different strategies of vector design (Ozturk, et al., 2006), with the most common consisting of the use of two

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Metabolic and Cell Engineering of Mammalian Cells vectors, one for each chain, which are co-transfected into the cells (Costa, et al., 2010). Their relative expression is controlled by the proportion of each gene used in the co-transfection mixture. This method cannot be control accurately is considered by Costa et al to be the least efficient to obtain a balanced expression of both chains, since the site of integration has a major effect on recombinant gene expression (Costa, et al., 2010). An alternative method is the use of a single vector using two identical promoters, one for each chain but this also introduces a risk of recombination events. A third method offers a more accurate control of the relative expression of multiple genes across a transfected cell pool, a single vector containing multiple compatible inducible promoters like cyclin E, cdc2, cyclin B1 and aurora A promoters, with each gene under the control of one independent promoter, can also be used (Costa, et al., 2010). This allows the introduction of an equal amount of different genes into each cell therefore changing the concentration of the corresponding chemical inducers. It is possible then to alter simultaneously the expression level of different genes (Costa, et al., 2010).

2.2.3. Transfection Methods

The introduction of the desired product gene into the host mammalian cell can be performed by a range of different methods and systems, in either a transient or stable way. As always the choice of which method is again dependent on the application required to produced your desired product, as well as economic and technical factors. The production of monoclonal anti bodies in mammalian cells for clinical or commercial purposes is usually done by stable transfection (Costa, et al., 2010). Stable transfection technology allows the user to develop a continuous expression of the product from mammalian cells over prolonged periods of time, thereby increase the production time and the delivery of larger quantities of therapeutic protein from the cells. A stable transfection is carried out by the integration of the DNA of the product gene into the genome of the host cell. To guarantee the integration, a marker gene is usually transfected in conjunction with the gene of interest, and the transfected cells producing the monoclonal anti body can be easily identified and selected by the characteristic provided by the product of the gene marker. This is, however, a labor-intensive and time-consuming step that involves considerable investment of resources/equipment (Costa, et al., 2010). A quicker and more economical approach for monoclonal antibody production is desired and can be provided by transient transfection. Transient transfection, although very similar to stable transfection can be distinguished by the elimination of the steps of identification and selection of cells that have integrated the plasmid into the genome in transient transfection (Costa, et al., 2010). This method provides a faster process for identifying monoclonal antibody-producing cells. Transient transfection does have a major drawback, since the product DNA is replicated as an extrachromosomal unit; the expression ability is rapidly lost, therefore only allowing the cell produce small quantities of monoclonal antibodies. Transient expression systems are not suitable for large-scale production but are very useful for high throughput screening in drug discovery processes, in vivo evaluation and early product analysis (Ozturk, et al., 2006). Other advantages of transient transfection is that it can be used with several cell lines such as, COS, HEK-293, CHO and BHK (Costa, et al., 2010). Due to the difference between stable and transient systems in relation to plasmid insertion into the genome, the factors that affect the overall expression level of the transfected cells also differ. For transient systems, the efficiency of transfection is one of the most important factors, consisting in the percentage of cells taking-up Paul S McCarthy Page 7 of 13 10274731

Metabolic and Cell Engineering of Mammalian Cells and expressing DNA. On the other hand, in stable systems, the frequency of DNA integration into the chromosome and the position of integration become more important. Nevertheless, for both systems, the expression levels are strongly affected by the strength of the promoter driving the expression of the product gene (Costa, et al., 2010).

2.2.4. Markers
The most commonly used marker genes are the dihydrofolate reductase (DHFR) an the glutamine d synthetase (GS).The first method is DHFR expression system, which is commonly used with CHO cells. The method is based on the dhfr gene coding for the DHFR enzyme, which is involved in nucleotide metabolism of catalyzing the conversion of dihydrofolate to tetrahydrofolate (Iwakura, et al., 1992). In this system, the selective advantage given to the transfected cells is the resistance to geneticin. Due to this resistance we can grow our culture of cells ells in a medium lacking hypoxanthine and thymidine (H/T) and containing geneticin, the cells that survive contains the marker. Furthermore, this system allows a process of ge amplification that allows the cell ne production capacity to increase by using methotrexate (MTX) (Costa, et al., 2010). Methotrexate is a drug that inhibits the DHFR enzyme, by increasing the concentration of MTX the transfected cells containing the dhfr gene will have to increase their capacity for DHFR synthesis therefore increasing the requirement of the dhfr gene in order to survive the increase of concentration. If our gene of interested is located along with the dhfr gene, then the production capacity of our cells increases. GS system is based on glutamine metabolism that is carried out in mammalian cells; the formation of glutamins in cells is through an enzymatic pathway of biosynthesis from glutamate and ammonium using the GS enzyme. Glutamins is essential for cell growth and without glutamine in the growth medium; this GS enzyme will not be able to produce glutamins for mammalian cells in culture (Costa, et al., 2010). Since some cell lines, such as mouse myeloma lines, do not express sufficient GS to survive, it is possible to use a transfected GS gene as a selectable marker by permitting growth in a glutamine-free medium (Costa, et al., 2010). Cell lines that express sufficient GS to survive can also be used with the GS system by using methionine sulphoximine (MTS), which is an inhibitor of the endogenous GS activity. Therefore only the transfectants that show additional GS activity by having the GS gene, are able to survive. MTS inhibitor also allows gene amplification using increasing levels of this toxic drug, similar to the principle used in the DHFR system with MTX. Other advantages of the GS system is the amount of time it saves over the DHFR system during development, and requires fewer copies of the recombinant gene per cell, allowing a faster selection of high-producing cell lines (Costa, et al., 2010).

2.2.5. DNA delivery systems

The most crucial element or step in cell engineering is the introduction of the gene of interest and the marker gene in stable transfection into the cells. There are number of different delivery systems available but the most preferred method is through non-viral gene transfer These methods include calcium phosphate precipitation, electroporation, lipofection and polymer-mediated gene transfer.

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Metabolic and Cell Engineering of Mammalian Cells

2.2.5.1. Calcium-phosphate Precipitation

Calcium phosphate precipitation is one of the most common methods for transferring plasmid DNA into cells to generate recombinant cell lines. This method is based on the formation of a fine DNA precipitate or complex that enters mammalian cells via an endocytic vesicle. Calcium-phosphate is quite inexpensive and works for a quite a large range of cell types (Ozturk, et al., 2006). But the major disadvantages are that its efficiency and reproducibility are quite low, and several studes have been carried out to improve the methodology. These include post-transfection treatments with dimethyl sulfoxide (DMSO), glycerol or chloroquine (Costa, et al., 2010),in order to support the transfer of a high plasmid copy number into cells, to increase transient and stable monoclonal anti body production.

2.2.5.2. Electroporation.
Electroporation is a simple and rapid method used for gene delivery into cells, the method is carried out my using a pulsed electric field to disrupts the voltage gradient across the plasma membrane, thereby forming reversible pores to allow the entry of DNA into the cell (Costa, et al., 2010). Though it is less cell type specific than other transfection methods, but considerations such as peak voltage and fall time of the discharge waveform need to be optimized for each cell type and use (Ozturk, et al., 2006). Another disadvantage of this method is lower post-transfection cell viability, consequently, this method is more commonly used in small assays that require rapid and low levels of production.

2.2.5.3. Lipofection and polyfection.

Lipofection and polyfection are the most recent, and probably most simple, transfect ion methodologies for gene delivery in a diversity of cells (Ozturk, et al., 2006). Lipofection is the cationic lipid-mediated gene transfer into the cell, this works by cationic liposomes forming a complex with the negatively charged DNA. This method can be performed in the presence or absence of se rum, with little or no toxicity, as well as with attached and suspended cells (Costa, et al., 2010). It is important to note that the amount of DNA and lipid used as well as the ratios between them affect transfection efficiencies and therefore still need to be optimized (Costa, et al., 2010). Polyfection is the use of cationic polymer such as polycation polyethylenimine, PEI and dendrimers to transfer our gene of interest into our chosen cell. The method works by interacting with DNA by forming a polyplex that protects DNA from degradation before reaching the cell nucleus. This method can be used for gene delivery in serum-free suspension cultures. As with lipofection, the optimal protocol needs to be optimized for each cell culture (Costa, et al., 2010).

2.2.6. Cell Selection

Once the cells are transfected they are subjected to a series of screening or selection test. These tests cover everything from growth recovery after transfection, through single-cell cloning, Paul S McCarthy Page 9 of 13 10274731

Metabolic and Cell Engineering of Mammalian Cells amplification, suspension and serum-free adaptation, until final clone selection. After transfection, monoclonal antibody producing cells are selected by meeting and surviving specific culture conditions that only allow survival and growth of cell clones expressing the marker gene product of our choice. These clones are transferred as single cells to a second cultivation vessel, where the cultures are expanded to produce clonal populations. Once the clones are evaluated in terms of growth and product titer, and the highest producers are selected. These cells are then subjected to another round of cultivation and analysis, to confirm the levels of productivity form that cell line are maintained (Costa, et al., 2010).

3. Metabolic Engineering
Another focus of genetic manipulation has been metabolic engineering as a way to indirectly increase cell growth and volumetric production, through the inhibition of the accumulation of toxic metabolic by-products, such as lactate and ammonia. Also to deter cells from initiating apoptosis, this is where cells can commit suicide.

3.1.

Apoptosis

Apoptosis is where the cells use signalling pathways to activate one of the following pathways, caspases, aspartate-specific and cystine-dependent proteases. Study has shown that protein such as IAPs or Bcl-2 family members are used to regulate apoptosis in the cell. Studies have also suggested that apoptosis is the predominant form of cell death in mammalian cell batchs and that apoptotic cells have been known to start in late exponential growth phase in hybridoma, myeloma, CHO and BHK cells (Kaufmann, et al., 2003). Kaufmann et al decribes the emergences of antiapoptosis engineering, which is aimed at reducing programmed cell death in production cultures by targeted molecular interventations in key apoptosis regulatory pathways (Kaufmann, et al., 2003). Studies that have been carried out so far are based on the overexpression of anti-apoptotic genes of the bcl-2 family of proteins.

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Metabolic and Cell Engineering of Mammalian Cells

Figure 1: Central components of the mammalian cell death manchinery. Programmed cell death or apoptosis is an evolutionary conserved process by which organisms remove damaged or superfluous cells. At the core of apoptosis execution as caspases, a family of aspartate -specific, cysteine-dependent proteases. Caspases function in a cascade in which procaspases are activated by upstream death signals. These signal include cytokine binding to receptors such as a members of the TNF receptor family and release of cytochrome C or DIABLO/Smac form the mitcohondria. Antiapoptotic members of Bcl-2 family of proteins can protect cells from various death stimuli acting on upstream death signals molecules. Inhibitors of apoptosis proteins (IAPs) such as XIAP bind and antagonize processed caspase 3 and 9. Arrows indicate protein targets for anti apoptsis engineering approaches.

3.2.

Engineering cells for hypothermic growth

Low temperature cultivation is a simple and very effective method of controlling cell proliferation, t was observed in studies that mammalian cells growing between 27C - 32C have a low specific growth rate, but maintain a high viability and reduced contamination by endogenous cell proteins (Costa, et al., 2010). But low temperature cultivation has been associated with increases in specific productivity, but this increase in specific productivity does increase the volumetric productivity, due to the reduced cell growth rates. To improve product production at low temperature a number of strategies have been determined. These strategies focus on alleviating the growth suppression at low temperature and include down-regulation of cold inducible RNA-binding protein (CIRP) with the intent to increase growth properties at hypothermia. Even with these strategies there is no increase in cell productivity even though there is an increase in cell growth at low temperatures. Alternatively, a biphasic process, where cells are first cultivated at 37C in the growth phase for high Paul S McCarthy Page 11 of 13 10274731

Metabolic and Cell Engineering of Mammalian Cells growth rates followed by a temperature shift to low culture temperature in the production phase for high productivity may be used to increase the volumetric productivity (Costa, et al., 2010)

3.3.

Glycosylation

Glycosylation variation, which can impact in vivo monoclonal anti body functions and stability, is one of the most sensitive quality-related attributes and it is vital in therapeutic protein product to ensure that our end product is at its highest quality. Cell culture factors such as host cell selection and protein specific features influence the glycosylation pathway and can lead to the formation of proteins with variable functionality (Costa, et al., 2010). In an attempt to achieve the required level of glycosylation and to improve effector functions, such as antibody-dependent cell mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), recent studies have focused on glycosylation engineering with specific manipulation of oligosaccharide structures (Andersen, et al., 2002). Glycosylation can be achieved by the over expression of appropriate glycosyltransferases, by improving glycan quality or by increasing homogeneity of native structures, or introducing non-host cell residues to specialize glycan quality and function (Costa, et al., 2010). Examples include the over expression of galactosyltransferase by an increase in the galactose levels sialyltransferase by an , increase in the sialic acid levels and N-acetylglucosaminyltransferase III, which is an increase the fraction of bisecting N-acetylglucosamine residues. The over expression of Nacetylglucosaminyltransferase III (GNTIII) can be achieved by transfecting cells with the GNTIII enzyme and results in nonfucosylated monoclonal (Costa, et al., 2010). Nonfucosylated monoclonal antibodies can also be obtained using cells with a-1,6-fucosyltransferase gene (FUT 8). Another approach to glycosylation is to introduce sialic acid in an a-2,6 linkage to glycoproteins synthesized by CHO and BHK cells. These glycoproteins lack the specific sialyltransferase responsible for this transfer (Costa, et al., 2010).

Bibliography
Cell Culture Technology for Pharmaceutical and Cell based Therapies [Book] / auth. Ozturk S S and Hu W s. - New York : CRC Press, 2006. Cell Engineering: Cell Line Development [Book] / auth. Al-Rubeai Mohamed. - [s.l.] : Springer, 2010. - Vol. 6.

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Metabolic and Cell Engineering of Mammalian Cells Dihydrofolate reductase gene as a versatile expression marker [Journal] / auth. Iwakura M and Tanaka T. - Ibaraki : Journal of Biochemistry, 1992. - 1 : Vol. 111. Expression Engineering - The IE2 Promoter/Enhancer from Mouse CMV [Book Section] / auth. Imhof Markus O [et al.] // Cell Engineering: Cell Line Development / book auth. Al-Rubeai Mohammed. - [s.l.] : Speinger, 2009. - Vol. 6. Fed-batch bioreactor process scale up from 3l to 2500l scale for monoclonal antibody produciton form cell culture [Journal] / auth. Yang J D [et al.]. - [s.l.] : Biotechnology Bioengineering , 2007. - Vol. 98. Genetically engineering mammalian cell lines for increased viability and productivity [Journal] / auth. Mosser Dick D and Massie Bernard. - Quebec : Biotechnology Advances, 1994. - 2 : Vol. 12. Guidlines to cell engineering for monoclonal antibody production [Journal] / auth. Costa Rita A [et al.]. - Braga : European Journal of Pharmaceutics and Biopharmaceutics, 2010. - Vol. 74. Identification of anto-repressor elements that confer high and stable protein production in mammalian cells [Journal] / auth. Kwaks T H.J [et al.]. - [s.l.] : Nature Biotechnology, 2003. - Vol. 21. Metabolic engineering of mammalian cells for higher protein yield [Book Section] / auth. Kaufmann Hitto and Fussenegger Martin // Gene Transfer and Expression in Mammalian Cells / book auth. Makrides Savvas. - [s.l.] : Elsvier, 2003. Production of monoclonal antibodies in COS and CHO cells [Journal] / auth. Trill J.J, Shatzman A R and Ganquly S. - [s.l.] : Current Opinion in Biotechnology, 1995. - Vol. 6. Production of recombinant protein theraputics in cultivated mammalian cell [Journal] / auth. Wurm F M. - [s.l.] : Naturel Biotechnology, 2004. - Vol. 22. Recombinant protein expression for therapeutic applications [Journal] / auth. Andersen D C and Krummen L. - [s.l.] : Current Opinion in Biotechnology, 2002. - Vol. 13. The construction of a mammalian transfection vector for expression of cytosine-5 specific DNA methyltransferase gene M.Msp1 in cultured cells [Journal] / auth. Ozdemir O. - [s.l.] : Turkish Journal of Biology , 1998. - Vol. 22. Uncoupling of Cell Growth and Proliferation Results in Enhancement of Productivity in p21CIP1Arrested CHO Cells [Journal] / auth. Bi Jing-Xiu, Shuttleworth John and Al-Rubeai Mohamed. - [s.l.] : Biotechnology and Bioengineering , 2004. - 7 : Vol. 85.

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