Drug Production in Cell Cultures and Bioreactor

Read this article to learn about the prospects of drug production in cell cultures and bioreactors.

Introduction:

Plant cell and callus cultures have been extensively used in the past three decades to explore the possibility of producing useful secondary metabolites through biotechnological methods.

These empirical approaches generated enormous data about the plant species producing secondary metabolites in culture, physicochemical factors like light, temperature, plant growth regulators and nutrients affecting the production and lastly, selection of cells with high yield of secondary metabolites. However, all the cultures investigated were not commercially viable systems.

Therefore, two way approaches was made:

(1) To generate the basic know-how for the production of secondary metabolites, and

(2) To develop technology for specific product using plants like Catharanthus roseus, Berberis, Coptis and Panax etc., as a model system. Investigations were focused to develop technology for these plants and also to better understand the process as a whole. Plant cell in the agitated liquid medium produces secondary metabolites characteristics of the parent plant, a process governed by several genes (i.e., genetically controlled) and hence it is a multi-steps reaction.

To produce secondary metabolites at commercial level there are certain pre­requisites like- high demand, high product cost, and availability of uniform raw material without interruption. Technology should be cost effective otherwise the production will not be commercially viable. Only a few selected compounds can fulfill these criteria because of high production cost. It is imperative that the production cost can be reduced by increasing the yield of secondary metabolites by improving the bioreactor technology to reduce the capital cost of inputs.

Serious attempts are being made to increase the yield by manipulating the biosynthetic pathways, removing the limiting factors barriers, and incorporating the techniques of genetic engineering. Here, we are presenting the details of bioreactor system and various cultures used to produce secondary metabolites in these bioreactors, towards developing technology for the industrial-level production. Application of plant cell tissue or organ culture for the production of secondary metabolites was originated in 1947, when James first reported the occurrence of alkaloids in meristem culture of solanaceous plants.

Scale-up (process at large-scale as compared to small-scale process in laboratory) problems are common in all kinds of engineering sciences. The classical processes of the fermentation industry have been developed along the time with trial and error method.

Bioreactors are advantageous then shake flasks as they provide better control on the system (i.e., pH, dissolved gas concentration, cell growth, etc.) there are examples of cultivation of plant cells in large fermenter demonstrating feasibility of the technology for the scale-up production of secondary metabolites. It is imperative that a bioreactor is used when basic studies related to optimization of product yield have been completed. In most cases, a growth medium and a production medium are used to obtain maximum yield of the secondary metabolite.

Tulecke and Nickel (1959) successfully developed a 10L system in a simple carboy for the cultivation of plant cells. Noguchi and co-workers (1977) cultivated Nicotiana tabacum cell suspensions in a 20,000 L stirred tank reactor while Schiel and Berlin (1987) studied Catharanthus roseus cells in a 5,000 L stirred tank reactor. The first commercial process used Lithospermum erythrorhizon cell suspensions grown in stirred tank reactors of 200 and 750L.

Various published results show that plant cell cultures can be grown in the microbial type bioreactors, thus suggesting the use of existing fermentation facilities and conventional equipment for industrial production of plant products using the cell culture technology. In the contrary to above, Archanbault et. al. (1998) suggested that specifically designed and operated bioreactors might be more appropriate for plant cell cultures. It is worth mentioning that different types of bioreactor designs and processes are currently in use for the production of secondary metabolites (Table 28.1).

Bioreactor Process:

Bioreactor is a large culture vessel made up of glass for use at laboratory-scale (up to 10L) but large-scale bioreactors are made up of stainless steel. Laboratory-scale bioreactors can be designed and fabricated in the laboratory or can be purchased as a functional unit (Fig. 28.1). The commercially available systems are sophisticated equipment fitted with microprocessor control unit (or a computer) to control the pH, dissolved oxygen, gas flow rate, agitation speed, nutrients, and temperature inside the vessel and cell density for the optimal growth and/or the production.

All the units have a culture vessel and a control unit. The details of a typical stirred type bioreactor are given in the figure. 28.2. There are different types of stirrers available to suit the requirement for tissue shear pressure and effective agitation. The modern bioreactors are fitted with various sensors like pH, temperature, foaming and nutrient concentrations. The control unit records the changes in the composition of the medium and then transfers the desired chemical/nutrient to adjust the medium according to set parameters.

Samples are taken, at time intervals to study the time course of the growth, with the help of sample tubes fitted with sterilized membrane filter and syringe to create a suction. For sterilization, small reactors are autoclaved while commercial scale reactors are sterilized in situ by passing steam at appropriate pressure.

Due care should be taken to sterilize the system according to its volume. Various kinds of bioreactors have been designed depending upon the requirements and the systems, However, all the bioreactors require a cooling unit (cryobath with water circulating pump) to circulate cold water (being a tropical country, in temperate countries, tape water is quite cold and can serve the purpose), a membrane type air pressure pump to supply oil-free air at a desired pressure, an autoclave of desired dimensions to sterilize the unit, disposable or autoclavable air filters, and autoclavable tubing’s to connect the system besides general facility for tissue and cell culture. A sufficient amount of stock of cell culture is required to inoculate the bioreactor.

This system is used to develop technology for the industrial production of useful secondary metabolites or for the production of large number of plantlets using somatic embryogenesis. Depending upon these factors for the accumulation/release of secondary products, different process modes are used viz., batch culture, fed batch culture, two-stage batch culture, and continuous chemostat type cultures. In batch cultures vessels are inoculated and harvested after growth period.

In fed-batch cultures, the cultures are fed once or twice with additional nutrient supply during the growth period. Additional supply of nutrients boosts the production of metabolites. In continuous cultures, a balance is maintained between continuous supply of nutrients and harvest of cells and cultures and in steady state of growth.

Factors for Growth in Bioreactor:

Gas-Liquid Mass Transfer:

Maintaining a constant oxygen mass transfer coefficient (K_La) is a basis of scale-up of many bio-processes. The oxygen transfer requirements of cultured plant cells are low as compared to that of bacterial cultures because unlike microorganisms, plant cells have lower respiration rates. For instance, if cells which respires at a rate of 0.2 mM g^-1 h^-1 are to be grown to 10 gl ^-1 without allowing the dissolved oxygen concentration to fall below 20% of saturation.

Plant cell culture in bioreactors typically requires KLa values between 10-30 h-1. Operation of bioreactor at higher KLa values results in poor cell growth or production of secondary metabolites. This may be due to either increased shear associated with high KLa conditions or due to enhanced CO₂ stripping from the medium. To maintain a constant dissolved oxygen level, bioreactors should be equipped with a dissolved oxygen probe to adjust aeration and agitation rate.

Shear:

Plant cells are shear sensitive. Shear refers to forces exerted on the surface of a body in a direction parallel to surface. This is contrasted to normal forces, which are exerted on a surface but, perpendicular to the surface.  Mathematically, shear can be described by the equation [τ = ηλ], where τ is the shear stress (a force per unit area), is the shear rate (a change in velocity across a distance), and η is a viscosity (a coefficient which describes the resistance to flow).

Earlier, efforts were made to describe the shear damage to eukaryotic cells growing in a mechanically agitated bioreactor system by calculating impeller tip speed by the equation [Tip speed = πND_i], where N is the impeller speed (rpm) and D_i is the impeller diameter. Sinskey et al. (1981) proposed that it may be better to use an integrated shear factor (ISF) to correlate shear damage to cells by the equation [ISF = πND_i/(D_t – D_i)] where, (D_t – D_i) is the measure of the distance between the impeller and the tank wall.

D_t is the tank diameter. ISF thus represents a pseudo-shear rate, which exists between the impeller tip and the vessel wall. Even in the absence of mechanical agitation, gas sparging (profuse bubbling of air/gas) can also exert shear.

Mixing:

Mixing of the dissolved nutrients of the culture medium is generally not been a problem in suspension cultures. But, large size of plant cells and especially the cell aggregates settle into dead zones or unmixed regions at the bottom of the bioreactor. Moreover, cells get adhere to the surface of the tank above the level of the medium and become deprived of nutrients. These exhibit a serious problem. Recently, there has been a trend towards using very high cell concentrations [packed cell volume (PCV of 90%)]. Under these conditions incomplete mixing can be a serious problem.