Introduction

The biopharmaceutical sector is largely based upon the application of techniques of molecular biology and genetic engineering for the manipulation and production of therapeutic macromol-ecules. The majority of approved biopharmaceuticals (described from Chapter 8 onwards) are proteins produced in engineered cell lines by recombinant means. Examples include the production of insulin in recombinant E. coli and recombinant S. cerevisiae, as well as the production of EPO in an engineered (Chinese hamster ovary) animal cell line.

Terms such as 'molecular biology', 'genetic engineering' and 'recombinant DNA (rDNA) technology' are sometimes used interchangeably and often mean slightly different things to different people. Molecular biology, in its broadest sense, describes the study of biology at a molecular level, but focuses in particular upon the structure, function and interaction/relationship between DNA, RNA and proteins. Genetic engineering, on the other hand, describes the process of manipulating genes (outside of a cell's/organism's normal reproductive process). It generally involves the isolation, manipulation and subsequent reintroduction of stretches of DNA into cells and is usually undertaken in order to confer on the recipient cell the ability to produce a specific protein, such as a biopharmaceutical. 'rDNA technology' is a term used interchangeably with 'genetic engineering'. rDNA is a piece of DNA artificially created in vitro which contains DNA (natural or synthetic) obtained from two or more sources.

When developing a new protein biopharmaceutical, one of the earliest actions undertaken entails identifying and isolating the gene (or complementary DNA (cDNA); see later) coding for the target protein, the generation of an appropriate piece of rDNA containing the protein's coding sequence and the introduction of this rDNA into an appropriate host cell such that the target protein is made in large quantities by that engineered cell. The drug development process and the cell types generally chosen to produce recombinant proteins are described in Chapters 4 and 5 respectively. This chapter aims to provide an introductory overview of the approaches and techniques used to isolate the target gene, generate an rDNA sequence and introduce it into an appropriate producer cell. Before we look at these techniques, however, we will briefly review the basic biology and structure of nucleic acids.

Pharmaceutical biotechnology: concepts and applications Gary Walsh © 2007 John Wiley & Sons, Ltd ISBN 978 0 470 01244 4 (HB) 978 0 470 01245 1 (PB)

Transcription

Reverse transcription

Translation

Protein

DNA RNA

replication replication

Figure 3.1 Schematic representation of the so-called central dogma of molecular biology. DNA replication is essential to the transmission of genetic information from one generation to the next in most life forms (i.e. in living forms whose genomes are DNA based). RNA replication is essential to the transmission of genetic information in the context of a small number of viruses whose genomes are RNA based. Transcription describes the copying of selected DNA sequences into RNA, and translation describes the conversion of the genetic information inherent in mRNA into a polypeptide of defined amino acid sequence. The process of reverse transcription is a central feature of certain viruses (retroviruses) containing an RNA-based genome which, as part of their life cycle, infect eukaryotic cells and convert their RNA-based genomes into a DNA-based one (see Box 14.1)

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