Proteins Macromolecules Recombinant protein

Biopharmaceuticals are protein macromolecules, usually prepared by recombinant DNA technology, which are used as therapeutics. This group includes replacement hormones such as insulin, cytokines such as interferons, and monoclonal antibodies. 

Enzyme immunoassay is widely used, both in competitive and non-competitive formats, for the bioanalysis of a broad range of low-molecular-weight compounds and macromolecules. Through the use of fluorogenic substrates and amplification systems such as avidin-biotin, the sensitivity of enzyme immunoassay has been developed to equal or exceed that of radioimmunoas-say.f ° The technique has found particularly wide applicability in the determination of new recombinant proteins, in demonstrating antibody responses to macromolecules, and in the measurement of biomarkers of disease, as well as in diagnostic medicine. 

This section focuses on the bioanaiyticai methods of validation applied to preclinical studies (nonhuman pharma-cology/toxicology) and human clinical pharmacological studies such as bioavailability and bioequivalence studies requiring pharmacokinetics (PK) evaluation [37, 38]. Inherently, differences exist between bioanaiyticai methods applied to preclinical and clinical development of small molecules versus recombinant proteins (macromolecules). Whereas... 

Since the number of recombinant protein drugs will substantially increase over the next decade, analytical as well as bioan-alytical methods for the characterization of such macromolecules will definitively gain in importance. Especially the vaKdation of bioanalytical methods is of key importance to allow an accurate description of protein drugs. This requires harmonized guidelines, which are not yet existing but seem to be on the way, to accelerate drug development. 

The purpose of this chapter is to review the principles of large zone size exclusion chromatography (large zone SEC) as applied to the study of self-association of macromolecules, specifically proteins. This selfassociation is not only of fundamental scientific interest, but also has practical consequences with respect to protein purification and to maintenance of biological function. The recent development of recombinant DNA technology has made available substantial amounts of sosie proteins, that were formerly available in very limited amounts if at all. These recombinant proteins must be obtained in a state that is fully biologically active. In some cases proteins are active as monomers, but in others the active protein exists as a dimer or an oligomer. Clearly it is of practical interest to understand the self-association of proteins under a variety of conditions. 

Nagapudi K, Brinkman WT, Leisen JE, Huang L, McMillan RA, Apkarian RP, Conticello VP, Chaikof EL (2002) Photomediated solid-state cross-linking of an elastin-mimetic recombinant protein polymer. Macromolecules 35, 1730-1737... 

Chimeric recombinant systems have also been developed, in an effort to combine the mechanical properties of silks with the biological features of other macromolecules. In one study, the consensus sequence of the major component of the dragline silk from Nephila clavipes was bioengineered into protein variants that incorporated RGD domains and expressed in E. coli. These recombinant proteins were able to maintain their beta-sheet forming abilities and were processable into films and fibers. Both RGD recombinant and recombinant silk without RGD supported human mesenchymal stem cell (hMSC) attachment and osteogenic differentiation. A separate study reported the fusion of domains from the major amplullate... 

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