A medium is a complex nutrient mixture that is used to grow and maintain the cells in vitro. A medium provides essential nutrients for cell growth and generation of the product. Among the nutrients that are necessary for cell growth are sugars, amino acids, fatty acids, vitamins, trace elements and possible other factors. In addition, a medium may contain phosphate buffer and antioxidants (Lambert and Birch, 1985; Freshney, 2005; Mather 1998). Most media also contain phenol red as a pH indicator (Mather, 1998).
2.3.1 Basal media
The most commonly used media for hybridoma cells are Eagle‟s minimal essential medium (MEM), Dulbecco‟s modification of Eagle‟s medium (DMEM), Ham‟s F12, Roswell Park Memorial Institute 1640 medium (RPMI 1640), Iscove‟s Modified Dulbecco medium (IMDM) and enhanced RPMI/DMEM/F12 medium (eRDF) in the volume ratio of 2:1:1. The composition of these media is given by Freshney (2005).
2.3.2 Serum-containing media
Conventionally, all culture media have been supplemented with 5 – 20% (vol/vol) serum. Calf, fetal bovine, horse and human sera have been used. Fetal calf serum (FCS; also known as fetal bovine serum, or FBS) is used most commonly. Serum provides the cells with an environment that attempts to reproduce the conditions found
17 Serum is a very complex and poorly characterized mixture of proteins and other molecules. Typically, serum contains protein such as albumin, insulin, transferrin and other growth factors that are lacking in the artificial basal media. Serum also acts as a pH buffer (Barnes and Sato, 1980; Maurer, 1986).
Although animal cells have been traditionally grown in serum-containing media, use of serum is associated with many problems. These include the following:
1. Batch-to-batch variability of serum because of variations in genetic, dietary and veterinary history of the animal leads to a variable production process (Shacter, 1989; Jayme and Smith, 2000; Freshney, 2005; van der Valk et al., 2010). 2. The poorly defined nature of serum means that it may contain unwanted
components that may interfere with the bioactivity of the product of interest (Barnes and Sato, 1980; Lambert and Birch, 1985; Maurer, 1986).
3. Presence of numerous protein and other undefined components in the serum can present major difficulties with downstream recovery of the product of interest (Jayme and Smith, 2000; Shacter, 1989; Velez et al., 1986; Blasey and Winzer, 1989; van der Valk et al., 2010).
4. Animal serum is potentially contaminated with etiologic agents such as viruses and mycoplasmas (Jayme and Smith, 2000; Freshney, 2005; Blasey and Winzer, 1989; Lambert and Birch, 1985; van der Valk et al., 2010). These agents are difficult to detect with certainty and, therefore, use of serum is undesirable especially when the product of the cell culture is to be used in vivo.
5. Serum is generally quite expensive and therefore best avoided to the extent possible (Jayme and Smith, 2000; Freshney, 2005; Velez et al., 1986; Kovář and Franěk, 1986).
In view of the problems mentioned above and the requirement for a consistent and defined medium for producing therapeutic and diagnostic products, extensive research has been carried out in attempts to totally or partially eliminate serum from cell culture media (Chang et al., 1980; Kovář and Franěk, 1986; Blasey and Winzer, 1989; Chua et al., 1994; Martial et al., 1995; Stoll et al., 1996; Radford et al., 1991; Yao et al., 2003;
Liu et al., 2001; Parampalli et al., 2007; Huang et al., 2007; Kim et al., 1998 and 1999;
18
2.3.3 Serum-free media
In an attempt to eliminate serum from media, Barnes and Sato (1980) found that both insulin and transferrin were stimulatory for every celltype that was studied by the authors. Some of the other cells required supplements in addition to the two already identified (Chang et al., 1980; Murakami et al., 1982; Kovář and Franěk, 1986; Chua et al., 1994; Murakami et al., 1997; Takenouchi and Sugahara, 2003; Müthing et al., 2003).
Using RPMI 1640 as a basal medium with the presence of insulin, transferrin and non-essential amino acids, Chang et al. (1980) successfully developed a serum-free medium for their hybridoma cell line (NS-19). They reported that the specific antibody production rate of their hybridoma in this serum-free medium was comparable to that in the serum-containing medium, although the maximum cell density achieved was greatly reduced. In another study, Kovář and Franěk (1986) supplemented RPMI 1640 with insulin, transferrin, ethanolamine, ascorbic acid, hydrocortisone and trace elements in culturing PTF-02 and T3-03 cells. This recipe was sufficient to support cell growth and produce an amount of MAb that was comparable to the level obtained in the serum- containing medium.
Several other basal media have been used to develop serum-free formulations for hybridoma growth. For example, Chua et al. (1994) used RPMI 1640, DMEM/F12 and eRDF supplemented with insulin, transferrin, ethanolamine, selenium and bovine serum albumin. The specific growth rates for the hybridoma cell line (2HG11) were lower in all three types of serum-free basal media tested as compared to their serum- supplemented counterparts. Nevertheless, MAb titer obtained in serum-free eRDF medium was the highest (~ 193 mg/L) among the media tested in static culture for about 7 days. Martial et al. (1995) employed IMDM/F12 (1:1) as the basal medium and supplemented it with L-glutamine, glucose, iron-saturated human transferrin, polyethylene glycol, ethanolamine, -mercaptoethanol, ascorbic acid, sodium selenite, essential amino acids, nonessential amino acids, bovine insulin and liposomes. They obtained cell density and MAb titer (25 mg/L) that were comparable to the values seen in the serum-containing medium during continuous culture of VO 208. It appears that a long list of supplements is not necessary for improving cell density and MAb
19 productivity, as clearly seen for the cases of Kovář and Franěk (1986) and Martial et al.
(1995).
Proprietary serum-free media such as HB101 (Franco et al., 1999), HB GRO (deZengotita et al., 2002a and 2002b), ASF103 and ASF104 (Terada et al., 2002) have also been used to culture hybridoma cells for producing monoclonal antibodies. The maximum cell density and MAb productivity achieved were comparable to the values observed with the corresponding serum-supplemented media. Different cell lines appear to require different recipes of serum-free media, and the recipe varies with the culture process and conditions (Maurer, 1986; van der Valk et al., 2010). Consequently, a case- by-case optimization of the formulation is important for developing serum-free media for use in large-scale culture.
2.3.4 Protein-free media
As stated in the Section 2.3.2, elimination of serum can possibly reduce the cost of large-scale cell culture. However, if serum is simply replaced with other expensive proteins, hormones and growth factors as mentioned in Section 2.3.3, the resulting serum-free medium may not reduce production costs (Lambert and Birch, 1985; Maurer, 1986; Shacter, 1989; Jayme and Smith, 2000). A long list of supplements further means an extended media preparation time (Shacter, 1989) and labor.
Moreover, supplementing a medium with proteins like insulin, transferrin and serum albumin can interfere with the recovery and purification of the secreted target protein (Nagira et al., 1995; Stoll et al., 1996; Jayme and Smith, 2000). This is particularly so when the target products are monoclonal antibodies that are usually produced in low concentrations compared to the concentrations of the protein additives such as insulin, transferrin and bovine serum albumin.
Serum and other animal-sourced protein are potentially contaminated with difficult-to-detect etiologic agents (Freshney, 2005; Shacter, 1989; Jayme and Smith, 2000; Hesse and Wagner, 2000; van der Valk et al., 2010). This is another reason for elimination of serum and other animal-derived proteins from culture media especially if
20 the objective is to produce a human therapeutic agent. This consideration is of course not of significance for in vitro diagnostic MAbs, but the high cost of serum is.
Blasey and Winzer (1989) worked towards eliminating bovine serum albumin from their medium. They attempted to replace serum‟s function as a fatty acid carrier with 0.1% (w/v) polyethylene glycol, PEG. The medium consisted of IMDM/F12 (1:1) supplemented with iron saturated human transferrin and bovine insulin. Initial experiments in batch culture were unsatisfactory as the MAb production ceased upon the cells reaching the stationary phase. A fed-batch strategy was more successful. A solution containing glucose, PEG and some MEM amino acid was used for feeding once the viable cell concentration had reached about 1 106 cells/mL. This caused a resumption of MAb production. As a result, a final cell density of 3 106 cells/mL and a final MAb titer of 187 mg/L were achieved. In another study (Chua et al., 1994), BSA was totally eliminated from the serum-free medium. This increased the maximum cell density attained, but the MAb level was not increased relative to control (Chua et al., 1994). Butler et al. (1999) found that linoleic acid could replace the function of bovine serum albumin in protecting cells under shear damaging conditions in the medium. The serum-free media of Blasey and Winzer (1989), Chua et al. (1994) and Butler et al.
(1999) did contain insulin and/or transferrin in their formulations.
Wong et al. (2004) successfully replaced the insulin in their serum-free medium with 1.5 mg/L of ZnSO4.7H2O. They were able to obtain 110 mg/L of antibody after 90
h of culture in an insulin-free Zn-supplemented medium. Iron compounds such as FeSO4 (Shinmoto et al., 1991) and ferric citrate (Kovář and Franěk, 1987; Nagira et al.,
1995; Franěk and Fussenegger, 2005) are able to successfully replace the iron-transport protein transferrin. Both the iron salts produced maximum cell concentration and MAb productivity results comparable to those obtained in transferrin-supplemented serum- free media. Clearly, development of a simple protein-free medium such as the one used by Wong et al. (2004) is possible without sacrificing productivity in at least some cell lines.
Several researchers have developed protein-free media by starting with a proprietary basal medium formulation such as FMX-Turbodoma. Stoll et al. (1996) supplemented this basal medium with gentamycin sulfate, glucose, glutamine, Pluronic
21 acid F-68 and several amino acids. Ducommun et al. (2001) supplemented FMX- Turbodoma with retinol acetate, ergocalciferol, D-alpha-tocopherol acetate, menadione sodium bisulfite, D-biotin, Ca-pantothenate, choline chloride, cyanocobalamine, folic acid, myo-inositol, niacinamide, pyridoxine HCl, riboflavine and thiamine HCl. In both cases, supplements significantly improved cell growth, prolonged the stationary phase before the onset of decline and increased the MAb production rate. In view of the above, in many cases it is possible to totally eliminate protein from culture media by providing appropriate supplements.
2.4 Factors affecting cell growth, cell metabolism and antibody production