Advances in our understanding of the molecular principles underlining both health and disease has revealed the existence of many regulatory polypeptides of significant medical potential. The fact that such polypeptides are produced naturally within the body only in minute quantities initially precluded their large-scale medical application. The development in the 1970s of the twin techniques of genetic engineering and hybridoma technology marked the birth of the modern biotech era. These techniques facilitate the large-scale production of virtually any protein, and proteins of medical interest produced by these methodologies have been coined ‘biopharmaceuticals’. More recent developments in biomedical research highlights the clinical potential of nucleic acid-based therapeutic agents. Gene therapy and anti-sense technology are likely to become a medical reality within a decade. The term ‘biopharmaceutical’ now also incorporates the polynucleotide sequences utilized for such purposes.

This book attempts to provide a balanced overview of the biopharmaceutical industry, not only in terms of categorizing the products currently available, but also illustrating how these drugs are produced and brought to market. Chapter 1 serves as an introduction to the topic, and also focuses upon several ‘traditional’ pharmaceutical substances isolated (initially at least) from biological sources. This serves as a backdrop for the remaining chapters, which focus almost exclusively upon recently developed biopharmaceutical products. The major emphasis is placed upon polypeptide-based therapeutic agents, while the potential of nucleic acid-based drugs is discussed in the final chapter.

In preparing the latest edition of this textbook, I highlight the latest developments within the sector, provide a greater focus upon actual commercial products thus far approved and how they are manufactured, and I include substantial new sections detailing biopharmaceutical drug delivery and how advances in genomics and proteomics will likely impact upon (bio)pharma-ceutical drug development.

The major target audience is that of advanced undergraduates or postgraduate students pursuing courses in relevant aspects of the biological sciences. The book should prove particularly interesting to students undertaking programmes in biotechnology, biochemistry, the pharmaceutical sciences, medicine or any related biomedical subject. A significant additional target audience are those already employed in the (bio)pharmaceutical sector, who wish to gain a better overview of the industry in which they work.

The successful completion of this text has been made possible by the assistance of several people to whom I owe a depth of gratitude. Chief amongst these is Sandy Lawson, who appears to be able to read my mind as well as my handwriting. Thank you to Nancy, my beautiful wife, who suffered most from my becoming a social recluse during the preparation of this text. Thank you, Nancy, for not carrying out your threat to burn the manuscript on various occasions, and for helping with the proof-reading. I am also very grateful to the staff of John Wiley and Sons Ltd for the professionalism and efficiency they exhibited while bringing this book through the publication process. The assistance of companies who provided information and photographs for inclusion in the text is also gratefully acknowledged, as is the cooperation of those publishers who granted me permission to include certain copyrighted material. Finally, a word of appreciation to all my colleagues at Limerick, who continue to make our university such a great place to work.

Pharmaceuticals, biologics and biopharmaceuticals

Pharmaceutical substances form the backbone of modern medicinal therapy. Most traditional pharmaceuticals are low molecular mass organic chemicals (Table 1.1). Although some (e.g. aspirin) were originally isolated from biological sources, most are now manufactured by direct chemical synthesis. Two types of manufacturing companies thus comprise the ‘traditional’ pharmaceutical sector; the chemical synthesis plants, which manufacture the raw chemical ingredients in bulk quantities, and the finished product pharmaceutical facilities, which purchase these raw bulk ingredients, formulate them into final pharmaceutical products, and supply these products to the end-user.

In addition to chemical-based drugs, a range of pharmaceutical substances (e.g. hormones and blood products) are produced by or extracted from biological sources. Such products, some major examples of which are listed in Table 1.2, may thus be described as products of biotechnology. In some instances, categorizing pharmaceuticals as products of biotechnology or chemical synthesis becomes somewhat artificial, e.g. certain semi-synthetic antibiotics are produced by chemical modification of natural antibiotics produced by fermentation technology.

The drug development process

In this chapter, the life history of a successful drug will be outlined (summarized in Figure 2.1). A number of different strategies are adopted by the pharmaceutical industry in their efforts to identify new drug products. These approaches range from random screening of a wide range of biological materials to knowledge-based drug identification. Once a potential new drug has been identified, it is then subjected to a range of tests (both in vitro and in animals) in order to characterize it in terms of its likely safety and effectiveness in treating its target disease.

After completing such pre-clinical trials, the developing company apply to the appropriate government-appointed agency (e.g. the FDA in the USA) for approval to commence clinical trials (i.e. to test the drug in humans). Clinical trials are required to prove that the drug is safe and effective when administered to human patients, and these trials may take 5 years or more to complete. Once the drug has been characterized, and perhaps early clinical work is under way, the drug is normally patented by the developing company, in order to ensure that it receives maximal commercial benefit from the discovery.

Upon completion of clinical trials, the developing company collates all the pre-clinical and clinical data they have generated, as well as additional pertinent information, e.g. details of the exact production process used to make the drug. They submit this information as a dossier (a multi-volume work) to the regulatory authorities. Regulatory scientific officers then assess the information provided and decide (largely on criteria of drug safety and efficacy) whether the drug should be approved for general medical use.

If marketing approval is granted, the company can sell the product from then on. As the drug has been patented, they will have no competition for a number of years at least. However, in order to sell the product, a manufacturing facility is required, and the company will also have to gain manufacturing approval from the regulatory authorities. In order to gain a manufacturing licence, a regulatory inspector will review the proposed manufacturing facility. The regulatory authority will only grant the company a manufacturing licence if they are satisfied that every aspect of the manufacturing process is conducive to consistently producing a safe and effective product.

Regulatory involvement does not end even at this point. Post-marketing surveillance is generally undertaken, with the company being obliged to report any subsequent drug-induced side effects/adverse reactions. The regulatory authority will also inspect the manufacturing facility from time to time in order to ensure that satisfactory manufacturing standards are maintained.

The drug manufacturing process

The manufacture of pharmaceutical substances is one of the most highly regulated and rigorously controlled manufacturing processes known. In order to gain a manufacturing licence, the producer must prove to the regulatory authorities that not only is the product itself safe and effective, but that all aspects of the proposed manufacturing process comply to the highest safety and quality standards. Various elements contribute to the safe manufacture of quality pharmaceutical products. These include:

. the design and layout of the manufacturing facility;

. raw materials utilized in the manufacturing process;

. the manufacturing process itself;

. the training and commitment of personnel involved in all aspects of the manufacturing operation

. the existence of a regulatory framework which assures the establishment and maintenance of

the highest quality standards with regard to all aspects of manufacturing.

This chapter aims to overview the manufacturing process. It concerns itself with four major themes: (a) a description of the infrastructure of a typical manufacturing facility, and some relevant operational issues — much of the detail presented in this section is equally applicable to facilities manufacturing non-biological-based pharmaceutical products; (b) sources of biophar-maceuticals; (c) upstream and downstream processing of biopharmaceutical products; and (d) analysis of the final biopharmaceutical product. Before delving into specific aspects of pharmaceutical manufacturing, various publications, such as international pharmacopoeias and guides to good manufacturing practice for medicinal products, will be discussed. These publications play a central role in establishing criteria which guarantee the consistent production of safe and effective drugs.

The cytokines —the interferon family

Cytokines are a diverse group of regulatory proteins or glycoproteins whose classification remains somewhat confusing, (Table 4.1). These molecules are normally produced in minute quantities by the body. They act as chemical communicators between various cells, inducing their effect by binding to specific cell surface receptors, thereby triggering various intracellular signal transduction events.

Most cytokines act upon or are produced by leukocytes (white blood cells), which constitute the immune and inflammatory systems (Box 4.1). They thus play a central role in regulating both immune and inflammatory function and related processes, such as haematopoiesis (the production of blood cells from haematopoietic stem cells in the adult bone marrow) and wound healing. Indeed, several immunosuppressive and anti-inflammatory drugs are now known to induce their biological effects by regulating the production of several cytokines.

The term ‘cytokine’ was first introduced in the mid-1970s. It was applied to polypeptide growth factors controlling the differentiation and regulation of cells of the immune system. The interferons (IFNs) and interleukins (ILs) represented the major polypeptide families classified as cytokines at that time. Additional classification terms were also introduced, including; lymphokines [cytokines such as interleukin-2 (IL-2) and interferon-g (IFN-g), produced by lymphocytes] and monokines [cytokines such as tumour necrosis factor-a (TNF-a) produced by monocytes]. However, classification on the basis of producing cell types also proved inappropriate, as most cytokines are produced by a range of cell types, e.g. both lymphocytes and monocytes produce IFN-a.

Initial classification of some cytokines was also undertaken on the basis of the specific biological activity by which the cytokine was first discovered, e.g. TNF exhibited cytotoxic effects on some cancer cell lines, colony stimulating factors (CSFs) promoted the growth in vitro of various leukocytes in clumps or colonies. This, too, proved an unsatisfactory classification mechanism, as it was subsequently shown that most cytokines display a range of biological.

Cytokines: interleukins and tumour necrosis factor

The interleukins (ILs) represent another large family of cytokines, with at least 25 different constituent members (IL-1 to IL-25) having been characterized thus far. Most of these polypeptide regulatory factors are glycosylated (a notable exception being IL-1) and display a molecular mass in the range 15–30 kDa. A few interleukins display a higher molecular mass, e.g. the heavily glycosylated, 40 kDa, IL-9.

Most of the interleukins are produced by a number of different cell types. At least 17 different cell types are capable of producing IL-1 (see Table 5.5), while IL-8 is produced by at least 10 distinct cell types. On the other hand, IL-2, -9 and -13 are produced only by T lymphocytes.

Most cells capable of synthesizing one IL are capable of synthesizing several, and many prominent producers of ILs are non-immune system cells (Table 5.1). Regulation of IL synthesis is exceedingly complex and only partially understood. In most instances, induction or repression of any one IL is prompted by numerous regulators — mostly additional cytokines, e.g. IL-1 promotes increased synthesis and release of IL-2 from activated T lymphocytes. It is highly unlikely that cells capable of synthesizing multiple ILs concurrently synthesize them all at high levels.

Nearly all ILs are soluble molecules (one form of IL-1 is cell-associated). They promote their biological response by binding to specific receptors on the surface of target cells. Most ILs exhibit paracrine activity (i.e. the target cells are in the immediate vicinity of the producer cells), while some display autocrine activity (e.g. IL-2 can stimulate the growth and differentiation of the cells that produce it). Other ILs display more systematic endocrine effects (e.g. some activities of IL-1).

The signal transduction mechanisms by which most ILs prompt their biological response are understood, in outline at least. In many cases, receptor binding is associated with intracellular tyrosine phosphorylation events. In other cases, serine and threonine residues of specific intracellular substrates are also phosphorylated. For some ILs, receptor binding triggers alternative signal transduction events, including promoting an increase in intracellular calcium concentration or inducing the hydrolysis of phosphotidylethanolamine with release of diacyl glycerol.

Haemopoietic growth factors

Blood consists of red and white cells which, along with platelets, are all suspended in plasma. All peripheral blood cells are derived from a single cell type: the stem cell (also known as a pluripotential, pluripotent or haemopoietic stem cell). These stem cells reside in the bone marrow, alongside additional cell types, including (marrow) stromal cells. Pluripotential stem cells have the capacity to undergo prolonged or indefinite self-renewal. They also have the potential to differentiate, thereby yielding the range of cells normally found in blood (Table 6.1). This process, by which a fraction of stem cells are continually ‘deciding’ to differentiate (thus continually producing new blood cells and platelets to replace aged cells), is known as haemopoiesis.

The study of the process of haemopoiesis is rendered difficult by the fact that it is extremely difficult to distinguish or separate individual stem cells from their products during the earlier stages of differentiation. However, a picture of the process of differentiation is now beginning to emerge (Figure 6.1). During the haemopoietic process, the stem cells differentiate, producing cells that become progressively more restricted in their choice of developmental options.

The production of many mature blood cells begins when a fraction of the stem cells differentiate, forming a specific cell type termed CFU-S (CFU refers to colony forming unit). These, in turn, differentiate yielding CFU-GEMM cells, a mixed CFU which has the potential to differentiate into a range of mature blood cell types, including granulocytes, monocytes, erythrocytes, platelets, eosinophils and basophils. Note that lymphocytes are not derived from the CFU-GEMM pathway, but differentiate via an alternative pathway from stem cells (Figure 6.1).

The details of haemopoiesis presented thus far prompt two very important questions. How is the correct balance between stem cell self-renewal and differentiation maintained? And what forces exist that regulate the process of differentiation? The answer to both questions, in particular the latter, is beginning to emerge in the form of a group of cytokines termed ‘haemopoietic growth factors’ (Table 6.2). This group includes:

. several (of the previously described) interleukins (ILs) that primarily affect production and differentiation of lymphocytes;

. colony stimulating factors (CSFs), which play a major role in the differentiation of stem- derived cells into neutrophils, macrophages, megakaryocytes (from which platelets are derived), eosinophils and basophils;

Hormones of therapeutic interest

Hormones are amongst the most important group of regulatory molecules produced by the body. Originally, the term ‘hormone’ was defined as a substance synthesized and released from a specific gland in the body which, by interacting with a receptor present in or on a distant sensitive cell, brought about a change in that target cell. Hormones travel to the target cell via the circulatory system. This describes what is now termed a ‘true endocrine hormone’.

At its loosest definition, some now consider a hormone to be any regulatory substance that carries a signal to generate some alteration at a cellular level. This embraces the concept of paracrine regulators (i.e. produced in the immediate vicinity of their target cells) and autocrine regulators (i.e. producer cell is also the target cell). Under such a broad definition, all cytokines, for example, could be considered hormones. The delineation between a cytokine and a hormone is already quite fuzzy using any definition.

True endocrine hormones, however, remain a fairly well-defined group. Virtually all of the hormones used therapeutically (discussed below) fit into this grouping. Examples include insulin, glucagon, growth hormone and the gonadotrophins.

Blood products and therapeutic enzymes

Blood and blood products constitute a major group of traditional biologics. The main components of blood are the red and white blood cells, along with platelets and the plasma in which these cellular elements are suspended. Whole blood remains in routine therapeutic use, as do red blood cell and platelet concentrates. A variety of therapeutically important blood proteins also continue to be purified from plasma. These include various clotting factors and immunoglobulins (immunoglobulins will be considered in the next chapter). Such blood products are summarized in Table 9.1.

Antibodies, vaccines and adjuvants

Few substances have had a greater positive impact upon human healthcare management than antibodies, vaccines and adjuvants. For most of this century, these immunological agents have enjoyed widespread medical application, predominantly for the treatment/prevention of infectious diseases. As a group, they are often referred to as ‘biologics’ (generally speaking, the term ‘biologic’ refers to vaccines, serum, toxins and medicinal products derived from human blood or plasma; Chapter 1).

Polyclonal antibody preparations have been used to induce passive immunity against a range of foreign (harmful) agents, while vaccines are used to efficiently and safely promote active immunization. Adjuvants are usually co-administered with the vaccine preparation, in order to enhance the immune response against the vaccine. The development of modern biotechnological methodology has had an enormous impact upon the therapeutic application of immunological agents, as discussed later. Monoclonal/engineered antibodies find a range of therapeutic uses, while many of the newer vaccine preparations are now produced by recombinant DNA technology.

Nucleic acid therapeutics

Throughout the 1980s and early 1990s, the term ‘biopharmaceutical’ had become virtually synonymous with ‘proteins of therapeutic use’ (Chapter 1). Nucleic acid-based biopharmaceu-ticals, however, harbour great potential — a potential which is likely to become a medical reality within this decade. Current developments in nucleic acid based-therapeutics centre around gene therapy and antisense technology. These technologies have the potential to revolutionize medical practice. Their full benefit, however, will accrue only after the satisfactory resolution of several technical difficulties currently impeding their routine medical application.

Despite all the hype, it is important to note that, by mid-2002 at least, only a single nucleic acid-based product has been approved for medical use (an antisense-based product, discussed later). No gene therapy-based product had been approved for general medical use by that time.

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