Expression systems, biopharmaceutical production

Although this chapter will focus on antibodies and fragments, the content clearly also applies to all biopharmaceuticals in the same way. Since the famous Kohler and Milstein [1] experiment in 1975, the success story of monoclonal antibodies was always linked to the development of appropriate expression systems and production technologies. 

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. 

Several key issues have to be addressed in the downstream processing of biopharmaceuticals regardless of the expression system. The removal of host cell proteins and nucleic acids, as well as other product- or process-related or adventitious contaminants, is laid down in the regulations and will not differ between the individual expression hosts. The identity, activity and stability of the end product has to be demonstrated regardless of the production system. The need for pharmaceutical quality assurance, validation of processes, analytical methods and cleaning procedures are essentially the same. 

Table 5.1 Expression systems which are/could potentially be used for the production of recombinant biopharmaceutical products...

OTHER EXPRESSION SYSTEMS FOR LARGE-SCALE PRODUCTION OF BIOPHARMACEUTICALS... 

The use of hairy roots for the production of biopharmaceuticals has been studied extensively and has been discussed in Chapter 1 of this book. To date, over 116 different plant species have been induced to produce hairy roots in culture (Guillon, 2006). Originally, an expression system was developed for protein production based on the natural secretion from roots of intact plants. In order to take up nutrients from the soil, interact with other soil organisms, and defend themselves against numerous pathogens, plant roots have developed sophisticated mechanisms based upon... 

The production of pharmaceutical proteins using hairy roots and rhizosecretion technology represents a safe and viable alternative to the use of whole plants for molecular pharming. As an example of the efficiency of this system, Medina-Bolivar and Cramer (2004) expressed the reporter protein GFP in tobacco hairy root cultures using a plastic sleeve bioreactor with a 5 L volume. Yields of 500 pg GFP/L after 21 days of incubation or 20% of total secreted protein were produced using this expression system, suggesting that rhizosecretion offers a promising production system for the production of biopharmaceuticals. 

Although numerous cell lines have been screened for their efficiency as a host system for recombinant protein production, only a few have shown favorable properties for the expression of biopharmaceuticals (Hauser, 1997). Regulatory and economic issues for large-scale production and the intended application of the recombinant protein (diagnosis, therapy, etc.) have to be carefully considered (Makrides and Prentice, 2003). Three mammalian cell lines are now commonly used by the pharmaceutical industry Chinese hamster ovary (CHO) cells, the murine myeloma SP2/0 and the NS0 cell line (see Table 3.1). These cell lines have been used to produce 11 of 21 therapeutic products approved from 1996 to 2000 (Chu and Robinson, 2001). 

The production of heterologous proteins for therapeutic use requires selection of the producer cell line, based on yield, monoclonality (for proteins), product quality, stability, and absence of contaminants like bacteria, molds, mycoplasmas, and viruses. Progress in the production of biopharmaceuticals by cell culture is due mainly to the use of diploid cells and continuous cell lines, together with the maintenance of cells by cryo-preservation. It is important to guarantee that the expression system chosen is able to generate the product in a consistent and economically feasible way (Levine and Castillo, 1999). 

Transgenic plants might also be used for production of recombinant proteins. While many of these systems are still early in the development stage, plants offer very robust and high-capacity system for biopharmaceutical production. Since plants cannot always properly modify proteins as mammalian cells, their utility may be limited. However, early studies suggest that plants may serve as useful hosts for production of vaccines and antibodies. For example, HepB surface antigen has been successfully expressed in potatoes [11], and clinical trials are underway with secretory antibodies (SIgAs), such as CaroRx , developed in plants [12]. 

Various types of validation generally required in biopharmaceutical manufacturing include process validation, facility and equipment validation, analytical method validation, software validation, cleaning validation and expression system characterization. Combined with other elements of cGMP, including lot release testing, raw material testing, vendor quality certifications, and vendor audits, the quality of product can be consistently assured. 

Mammalian cell culture is becoming increasingly important for the production of high-volume biopharmaceutical proteins. This is driving improvements in process efficiency. This chapter provides examples of improvements in both the creation of cell lines and in cell culture optimization, focusing particularly on experience with the glutamine synthetase (GS) expression system. 

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