Using the broadest definitions, a biopharmaceutical may be defined as any molecule intended for therapeutic, prophylactic or diagnostic use that was generated by a manufacturing process incorporating the use of living cells. This definition applies even if the therapeutic entity is thought to be identical in molecular structure to a naturally occurring substance. The most common biopharmaceuticals to date are proteins derived from recombinant DNA technologies and monoclonal antibodies resulting from hybridoma technology.
In simple terms, recombinant DNA technology is the manipulation of nucleic acids (DNA, RNA) in a host cell to produce a new protein sequence or amplify production of an endogenous protein considered to be of diagnostic or therapeutic value. The experimental conditions are optimized such that the desired protein is over-expressed in the host cell system of either prokaryotic or eukaryotic origin to attain high yields of the product. In hybridoma technology, an antibody producing B-lymphocyte is fused with a myeloma cell and the resulting hybridoma cells cloned. The purified product of a cloned hybridoma cell line is a monoclonal antibody directed at a specific antigenic determinant.
Advances in molecular biology, genetic engineering, process purifications, analytical chemistry and related disciplines have led to the production of large quantities of highly purified proteins facilitating drug development efforts. A number of these proteins such as insulin, human growth hormone (HGH), a-interferon, y-interferon, tissue plasminogen activator (TPA), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), Interleukin-2, erythropoietin, hepatitis B-vaccine and murine monoclonal antibody have already been approved by the Food and Drug Administration (FDA) for human use in the United States while a number of others are currently under active development as potential therapeutic agents.
In order for a drug to be approved for human use it is the responsibility of the manufacturer to demonstrate its safety and efficacy by means of well controlled preclinical animal studies and human clinical trials [1, 2], Once the inherent safety and efficacy of the intended pharmaceutical has been established, the manufacturer is then required to demonstrate the capability to consistently manufacture the intended drug product of a quality that is at the very minimum equivalent or preferably better than that used in the safety assessment and clinical evaluation phases of development. This necessitates the availability of methods that are capable of assessing the quality of the safety-assessment supplies and the clinical supplies. The potential molecular complexity of biopharmaceuticals has created an unprecedented opportunity for the advancement of analytical biotechnology as pharmaceutical analytical chemists strive to develop appropriate methodologies that can adequately characterize these molecules. This situation has facilitated improvement of some classical biochemical techniques such as slab gel electrophoresis and accelerated development of others such as tryptic mapping, carbohydrate analysis, bioassays, ELISA, mass spectrometry and capillary electrophoresis to meet the regulatory needs for drug approval.
The marketing of all pharmaceuticals in the United States is regulated by the Code of Federal Regulations for Drugs, 21 CFR, Chapter 1, Sub-chapters C and D. Biological products which include all vaccines, antibiotics, hormones, human blood or blood-derived products, immunoglobulin products, products containing intact cells, fungi, viruses or virus pseudotypes, proteins produced by cell culture or transgenic animals and animal venoms; synthetically produced allergenic products; and drugs used for bloodbanking and transfusion are further regulated by the Code of Federal Regulations for Biologies, 21 CFR, Chapter 1, Subchapter F. While the fundamental issues regarding safety and efficacy of biopharmaceuticals are essentially the same as those for conventional pharmaceutical agents, the complexity of biotechnology products has necessitated additional guidance regarding their manufacture and control. This need has been met in part by the collaborative efforts of the biopharmaceutical industry, academia and the regulatory agencies, resulting in the issuance of a number of Guidelines [3-5] and "Points to Consider" documents [6-8] to aid the industry in assuring the identity, purity, safety and potency of new drugs generated from recombinant DNA and hybridoma technologies.
More recently, there has been a major initiative to harmonize the technical requirements for the registration of pharmaceutical products in the United States, the European Union and Japan prompted by the need to streamline the drug development process, to minimize drug development costs and to accelerate drug approval. This has led to the development and issuance of a number of new guidelines as part of the tripartite harmonization initiatives by the International Conference on Harmonization (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. [4, 5] The six sponsors of the ICH are the US Food and Drug Administration, US Pharmaceutical Research and Manufacturers Association, the European Commission, the European Federation of Pharmaceutical Industry Associations, the Japanese Ministry of Health and Welfare, and the Japanese Pharmaceutical Manufacturers' Association. Many of the new or revised guidelines are still in the draft stages and are expected to be finalized and issued shortly. The ICH guidelines are discussed in more detail in Chapter 3 of this publication.
Every lot of a pharmaceutical product intended for human use must be assured of its safety, efficacy and purity. This is generally accomplished by application of a variety of techniques to meet the key analytical requirements of identity, purity, potency (biological activity), strength (concentration), safety and stability.
Minimum requirements for the identity tests are met by comparison of the property of the analyte to that of a pre-established, well-characterized reference standard. A combination of qualitative tests are often used to unequivocally establish the identity of a biopharmaceutical which may include molecular size/weight determination by size exclusion chromatography or gel electro-
phoresis, amino acid analysis, peptide mapping and protein sequencing. In addition to confirming the identity of the macromolecule these techniques also provide key information regarding the primary and secondary structure.
Purity analysis of a pharmaceutical protein is better described as impurity analysis as there is no single analytical technique that yields a purity value of a given sample. The approach is usually to identify and quantitate all known or potential impurities in the drug product from which an assessment of overall purity can be made.  The types of impurities that are found in protein pharmaceuticals and the analytical techniques commonly used to identify/quantitate them are shown in Table 9.1. Proteinaceous impurities may be introduced from a number of sources during the synthesis, isolation and purification phases of the manufacturing process. These include contaminating host cell proteins which may be copurified with the product; altered or inactive forms of the protein; products of the interaction of the intended protein with reagents used in isolation and purification; proteins added for growth promotion during the fermentation stage of the process and proteins that may have leached into the final product during purification by affinity chromatography.
Process related impurities, also known as non-adventitious impurities, are substances present in the raw materials, bulk drug substance or final drug product that are not considered to be active ingredients, additives or excipients. They can be innocuous in that they do not pose a health hazard or deleterious in that they may be considered a health or safety concern, particularly with respect to toxicity, carcinogenicity or immunogenicity. Deleterious impurities must be controlled within safe limits and their levels must be determined in every lot of drug product using appropriate analytical techniques. Contaminants, sometimes referred to as adventitious impurities are biological or chemical agents that may be accidentally introduced into the drug product. While these are difficult to control entirely, such events must be anticipated and appropriate broad spectrum, non-specific analytical methods such as LC with low wavelength detection should be incorporated into the drug product monograph such that these agents, if present, will be detected. Analytical methods should be developed to detect and quantitate impurities or related compounds that are considered to be endogenous to the process and would be expected to be present in the drug.
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