A central paradigm of the biopharmaceutical industry is to identify a natural protein having a desired biochemical activity and then use recombinant methods to produce that protein in sufficient quantities for biological testing and ultimately, for therapeutic use, usually as an injected drug. For proteins intended for use as human pharmaceuticals, a key experiment will be to test the protein for efficacy in an animal model of the human disease. Testing of these proteins, however, may be limited by factors other than their intrinsic biochemical activity. Poor solubility, stability, and pharmacokinetic half-life are common issues hampering preclinical and clinical testing of protein pharmaceuticals. In particular, low-molecular-weight proteins usually show rapid clearance in vivo, making it difficult to deliver a high-enough dose to achieve the desired biological effect. Although smaller molecular size may have advantages, for example, in penetration into solid tumors, for many injected therapeutics, prolonged half-life and infrequent dosing are often preferred.

Protein engineering can alter many functional properties of peptides and proteins and, combined with high-throughput screening or molecular diversity approaches, such as phage display, may often lead to dramatic alterations in binding affinity toward a target receptor or antigen. In recent years, protein engineering efforts have also turned to improvement of the pharmacokinetic properties of first- or second-generation polypeptide drugs, which are often limited in their pharmacokinetic half-life by glomerular filtration in the kidney (1-3). Monoclonal antibodies (MAbs) and other protein drugs may be modified with substitutions of methionine or asparagine residues that may be otherwise subject to oxidation or deamidation, respectively, in vitro or in vivo, to yield reduced functional activity over time. In addition, proteolytic sites, such as dibasic sequences (consecutive arginine or lysine residues), have been recognized as potential sources of in vivo instability and may be altered through protein engineering (4,5). In this chapter, we omit discussion of such protein-specific issues, which differ greatly even among therapeutic antibodies according to their diverse complimentarity-determining regions (CDRs), and focus instead on three general strategies for improving the pharmacokinetic half-lives of injected peptides and proteins (Fig. 1).

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