Biopharmaceutical Production in Cultured Plant Cells

Stefan Schillberg, Richard M. Twyman, and Rainer Fischer 

Copyright 2005 WILEY-VCH Verlag GmbH Co. KGaA, Weinheim ISBN 3-527-31184-X 

Many different plant-based expression systems are now available for the production of recombinant proteins. Those utiHz-ing whole plants have been extensively reviewed, and will not be discussed in detail here (see Refs. [5-8]). Other systems, based on cultured plant cells or organs, include hairy roots [9], shooty teratomas [10], 

Suspension cell cultures have been derived from a number of different plant species, including the widely-used laboratory model Arabidopsis thaliana [15], plants such as Catharanthus roseus and Taxus cus-pidata which are used to produce valuable secondary metaboHtes [16, 17], and important domestic crops such as tobacco, rice, alfalfa, tomato, and soybean [18-22]. Because cell lines from domestic crop species are well-characterized, they have been the most frequently used for recombinant protein production. The most popular cell hnes include those derived from the tobacco cultivars Bright Yellow 2 (BY-2) (Fig. 9.1) and Nicotiana tabacum 1 (NT-1) [2]. 

Recombinant Proteins Produced in Plant Cell Suspension Cultures 

---

In this review, we focus on the use of plant tissue culture to produce foreign proteins that have direct commercial or medical applications. The development of large-scale plant tissue culture systems for the production of biopharmaceutical proteins requires efficient, high-level expression of stable, biologically active products. To minimize the cost of protein recovery and purification, it is preferable that the expression system releases the product in a form that can be harvested from the culture medium. In addition, the relevant bioprocessing issues associated with bioreactor culture of plant cells and tissues must be addressed. 

As indicated in Table 2.1, most of the promoters used in plant tissue culture have been based on the constitutive cauliflower mosaic virus (CaMV) 35S promoter. In contrast, inducible promoters have the advantage of allowing foreign proteins to be expressed at a time that is most conducive to protein accumulation and stability. Although a considerable number of inducible promoters has been developed and used in plant culture applications, e.g. [32-37], the only one to be applied thus far for the production of biopharmaceutical proteins is the rice a-amylase promoter. This promoter controls the production of an a-amylase isozyme that is one of the most abundant proteins secreted from cultured rice cells after sucrose starvation. The rice a-amylase promoter has been used for expression of hGM-CSF [10], aranti-trypsin [12, 29, 38, 39] and human lysozyme [30]. 

Plant cell culture has been used extensively for the production of biopharmaceuticals. A few examples are illustrated here, and a more extensive list can be found in Table 6.4. Smith et al. (2002) employed both soybean and tobacco cell lines to produce a hepatitis virus surface antigen (HBsAg), to be used as a vaccine, in shaker flask cultures. The authors found that the titers of HBsAg in soybean cell culture were 65 pg/g fresh weight, and 10-fold lower in tobacco cell culture, resulting in productivities of 1 mg/L/d and 0.16 mg/L/d, respectively. These numbers correspond well with those found for yeast batch cultures (1.5 mg/L/d). 

As well as overcoming many of the inherent problems associated with agriculture, plant tissue culture also offers a number of advantages over conventional animal cell culture methods currently being applied to produce biopharmaceutical proteins commercially [8], As plant culture media are relatively simple in composition and do not contain proteins, the cost of the process raw materials is reduced and protein recovery from the medium is easier and cheaper compared with animal cell culture. In addition, as most plant pathogens are unable to infect humans, the risk of pathogenic infections being transferred from the cell culture via the product is also substantially reduced. 

---