## Info

Centroid

Centroid

Carbon source conc

Carbon source conc —

Fig. 4.4. (a) Simplex optimization for a pair of independent variables (with reflection), (b) Simplex optimization of pair of independent variables which has reached the optimum.

Fig. 4.4. (a) Simplex optimization for a pair of independent variables (with reflection), (b) Simplex optimization of pair of independent variables which has reached the optimum.

now used to design the next experiment. A new simplex (equilateral triangle) BCD is constructed opposite the worst response (i.e. A) using the existing vertices B and C. A line is drawn from A through the centroid (mid point) of BC. D (the next experiment) will be on this line and the sides BD and CD will be the same length as BC. This process of constructing the new simplex is described as reflection. Once the position of D is known, the concentrations of the carbon and nitrogen sources can be determined graphically, the experiment performed and the production of antibiotic assayed. Thus, a series of simplexes can be constructed moving in a crabwise way. The procedure is continued until the optimum is located. At this point the simplex begins to circle on its self, indicating the optimum concentration (Fig 4.4b; Greasham and Inamine, 1986). However, if a new vertex exhibits the lowest response, the simplex would reflect back on to the previous one, halting movement towards the optimum. In this case the new simplex is constructed opposite the second least desirable response using the method previously described.

If it is decided that the supposed optimum should be reached more rapidly then the distance z between the centroid and D may be increased (expanded) by a factor which is often 2. If the optimum is thought to have nearly been reached then the distance z may be decreased by a factor of 0.5 (contraction). This modified simplex optimization was first proposed by Nelder and Mead (1965) and has been discussed by Greasham and Inamine (1986).

The simplex method may also be used in small scale media development experiments to help identify the possible optimum concentration ranges to test in more extensive multifactorial experiments.

### Animal cell media

Mammalian cell lines have been cultured in vitro for 40 years. Initially, animal cells were required for vaccine manufacture but they are now also used in the production of monoclonal antibodies, interferon, etc. The media initially used for this purpose contained about 10% serum (foetal calf or calf) plus other organic and inorganic components. Since this pioneering work it has been possible to develop a range of serumfree media (Ham, 1965; Barnes and Sato, 1980). These media contain carbohydrates, amino acids, vitamins, nucleic acids, etc, dissolved in high purity water. Media costs are therefore considerably higher than those for microbial cells. At a 1000 dm3 scale the medium costs may account for 40% of the unit costs, and serum may be 80% of the medium cost (Wilkinson, 1987).

Serum

The serum is a very complex mixture containing approximately 1000 components including inorganic salts, amino acids, vitamins, carbon sources, hormones, growth factors, haemoglobin, albumin and other compounds (Brooks, 1975; Glassy et al., 1988). However, most of them do not appear to be needed for growth and differentiation of cell lines which have been tested (Barnes and Sato, 1980; Darfler and Insel, 1982).

Serum is used extensively in production media for animal cell culture to produce recombinant proteins and antibody based products for in vivo use in humans. At present the regulations governing the quality of serum which can be used for manufacturing processes vary considerably from country to country (Hodgson, 1993). However, FDA approval of a process will be essential to market a product in the USA and therefore regulate the quality of serum which can be used. Serum tested by approved laboratories should be free of bacterial, viral or BSE (bovine sporangiform encephalitis) contamination and other components should be within strictly defined limits. Serum of this standard is needed for the cell culture media which is used to maintain the cell culture stocks as well as the production media.

The cost of foetal calf serum, US\$190 dm"3 in Europe, makes serum free media attractive economic alternatives, but it would take a number of years to develop suitable serum free media. The absence of the many unutilized components in serum will also simplify purification of potential products produced in such media. However, these process changes would need approval by the FDA or other regulatory bodies before a product could be marketed using a modified process.

### Serum-free media supplements

The development of serum-free media was initiated by Ham (1965) who reduced the amount of serum in media and optimized other medium components and Sato (Barnes and Sato, 1980) who investigated a range of components to promote cell growth and differentiation. Some of the more important replacements in serum-free media are albumin, insulin, transferrin, ethanolamine, selenium and /3-mercaptoethanol (Glassy et al., 1988).

The advantages of removing serum from media include:

1. More consistent and definable medium composition to reduce batch variation.

2. Reduction in potential contamination to make sterility easier to achieve.

3. Potential cost savings because of cheaper replacement components.

4. Simplifying downstream processing because the total protein content of the medium has been reduced.

### Protein-free media

The elimination of proteins seems an attractive objective. However, the design of such media is difficult and their use may be very limited and not very cost effective. Hamilton and Ham (1977) demonstrated the growth of Chinese hamster cell lines in a protein-free medium formulated from amino acids, vitamins, organic compounds and inorganic salts. Other media have been developed by Cleveland et al. (1983) and Shive et al. (1986).

### Trace elements

The role of trace elements in medium formulation can be significant. Cultured cells normally require Fe, Zn, Cu, Se, Mn, Mo and V (Ham and McKeehan, 1975). These are often present as impurities in other media components. Cleveland et al. (1983) found that if the number of trace elements were increased, insulin, transferrin, albumin and liposomes were not needed in a serum-free hybridoma medium. They included Al, Ag, Ba, Br, Cd, Co, Cr, F, Ge, J, Rb , Zr, Si, Ni and Sn as well as those previously mentioned.

### Osmolality

The optimum range of osmotic pressure for growth is often quite narrow and varies with the type of cell and the species from which it was isolated. It may be necessary to adjust the concentration of NaCl when major additions are made to a medium.

The normal buffer system in tissue culture media is the COz-bicarbonate system. This is a weak buffering system and can be improved by the use of a zwitteri-

onic buffer such as Hepes, either in addition to or instead of the C02-bicarbonate buffer. Continuous p| | control is achieved by the addition of sodium bicarbonate or sodium hydroxide (with fast mixing) when too acid. The pH does not normally become too alkaline so acid additions are not required but provision may be made for C02 additions (Fleischaker, 1987).

### Non-nutritional media supplements

Sodium carboxy methyl cellulose may be added to media at 0.1% to help to minimize mechanical damage caused by the shear force generated by the stirrer impeller. The problems of foam formation and subsequent cell damage and losses can affect animal cell growth. Pluronic F-68 (polyglycol) can provide a protective effect to animal cells in stirred and sparged vessels. In media which are devoid of Pluronic F-68, cells may become more sensitive to direct bubble formation in the presence of an antifoam agent being used to su-press foam formation (Zhang et al., 1992).