In its broadest definition,biotechnology refers to the use of living organisms,including isolated mammalian cells,in the production of products having beneficial use.This definition would place alcohol,antibiotic production,and dairy processing,for example,within the scope of biotechnology.However,the current interest in biotechnology is primarily a result of two major advances.The first advance was the development of rDNAtechnology,which allowed the genes of one species to be transplanted into another species.Thus,gene coding for the expression of a desired protein (usually human)could be inserted into a host prokaryotic or eukaryotic cell in such a manner that the host cell would then express usable quantities of the desired protein.The second major advance was the development of techniques for producing large quantities of monoclonal antibodies (i.e.,antibodies arising from a single lymphocyte).

Biotechnology within the pharmaceutical industry generally refers either to the production of protein products using rDNAtechniques or to the production of monoclonal antibodies.Other technologies,such as transgenic animals and plants,gene therapy,and antisense DNA,may have potential implications for the pharmaceutical industry in the future but are not within the scope of this chapter.

The major steps in the application of rDNAtechnology for production of a desired protein are outlined in this section.The critical first step is identification of the protein that is to be produced,followed by the isolation of the gene of interest (i.e.,the DNAsequence coding for the desired protein).Once this gene is isolated and fully characterized,it is inserted into a suitable vector such as a plasmid,which is an extrachromosomal segment of DNAusually found in certain bacteria.The plasmid is then inserted into the host cell.Clones of the transformed host cell line are isolated,and those that produce the protein of interest in the desired quantities are preserved under suitable conditions as a cell bank.As manufacturing needs arise,the cloned cells can be scaled up in a fermentation or cell culture process to produce the protein product.

Although the rDNAprocess is more fully described elsewhere in this chapter,the following important points should be recognized.The vector (plasmid)generally contains a selectable marker that can be used to identify cells that contain this gene.This is in addition to the gene coding for the protein of interest and the regulatory nucleotide sequences necessary for plasmid replication and messenger RNA(mRNA)transcription (the first step in protein synthesis).Selection of the desired cells is simplified because only properly transformed cells containing the selectable marker gene will survive under the growth conditions used to identify and propagate the transformed cells.Typically,the bacterial and eukaryotic selectable markers may include both antibiotic resistance or genes that complement an auxotrophic host mutation.There are numerous examples of both types of markers in each system.

Significant differences exist in the rDNAproduction process between prokaryotic and eukaryotic cells.In general,bacterial cells express greater concentrations of protein product and require relatively simple media components.However,prokaryotic cells do not perform many important post-translational modifications such as glycosylation and,historically,it was not possible to express large proteins in E.coli.These limitations necessitate the use of eukaryotic cells in many cases.The production differences between eukaryotic and prokaryotic host cells have significant impacts that are reflected in the requirements for process validation,purification,and analytical methodology.These requirements are addressed later in this chapter.

Antibodies are proteins produced by differentiated Blymphocytes.Each lymphocyte produces an antibody of defined specificity (i.e.,the antibody molecule recognizes a specific site or epitope on the antigen).Antibodies that are produced in immunized animals are formed from many different clones of Blymphocytes;hence,the name polyclonal antibodies.Because the harvest of blood from these animals,by definition,results in polyclonal antibody mixtures,the antisera have multiple epitope recognition sites with a wide variety of binding constants (avidity)and therefore vary from lot to lot.Antibodies that are produced by immortalized cell lines (hybridomas)derived from single Bcells are referred to as monoclonal antibodies.The harvest of these cultures leads to an antibody of specific epitope recognition with a homogeneous binding constant.

Blymphocytes have a finite life span in culture and have to be immortalized to enable continuous monoclonal antibody production.At present,the most common procedure is through chemically-induced fusion of a mouse spleen cell with a mouse myeloma cell.The resultant mouse-mouse hybridoma cell inherits from the myeloma cell the ability to replicate continuously in culture and inherits from the spleen cell the ability to produce the desired monoclonal antibody.Cell banks of the hybridoma cell line can be used to produce a continuous supply of the monoclonal antibody,either in vivo (i.e.,by injection into mice and subsequent collection of the ascites fluid),or in vitro (i.e.,by conventional cell culture techniques).It should be mentioned that recent advances in molecular genetics have led to the development of transfectomas and E.coli-and bacteriophage-based production schemes that may offer advantages for future production of monoclonal antibodies.

Process validation,purification,and analytical considerations for monoclonal antibodies are conceptually similar to those for rDNAproducts.This is because both types of products are proteins and therefore require similar handling and assay procedures.Because monoclonal antibodies are the products of immortalized cell lines,there is concern that potential viral nucleic acid contaminants be effectively excluded or inactivated by the manufacturing processes,just as for recombinant products of continuous cell lines.

Commercial applications of monoclonal antibodies include both diagnostic and therapeutic uses.In some cases,the monoclonal antibody is coupled to another substance (e.g.,an oncolytic agent,radionuclide,toxin),with the resultant antibody conjugate being the final product of interest.In this case,both the antibody intermediate and the final product require extensive process development and analytical characterization.

For the purposes of this chapter,the scope of biotechnology will be confined to rDNAand monoclonal antibody pharmaceutical products.