Here ya go:
Method for determining the biological activity of recombinant human growth hormone.
Boris Y Zaslavsky
Department of Physiology and Biophysics. Cornell University
Recombinant DNA (rDNA) technology has led to the development of new protein-based drugs that are gaining worldwide regulatory approval. Human growth hormone, human insulin, ?- and ?-interferons, and erythropoietin are just a few examples of approved rDNA-derived biopharmaceuticals.
The biological effects, purity, and potency of a drug is governed by the chemical structure of the drug for both traditional drugs and biopharmaceuticals. Standard analytical methodologies used for structural analysis of conventional drugs are, however, inadequate for complete characterization of protein-based products.
Two main reasons for this inadequacy are the large molecular size and conformational flexibility of protein-based drugs. The large molecular size hinders the possibility to detect, for example, replacement or chemical modification of a single amino acid residue or a change in a single glycosylation site. These alterations of the biomolecule structure, however, may lead to subtle changes of the molecule conformation resulting in significant changes in the pharmacological properties of the product.
Additionally, the wrong choice of manufacturing conditions or formulation may lead to improperly folded polypeptide chains which are biologically inactive. Hence, further methodologies capable of analysis of the protein conformation are needed.
Currently, the analysis of biopharmaceuticals relies heavily on the use of sophisticated methods for the demonstration of the structural identity, homogeneity and purity of the products. These methods include amino acid and carbohydrate analysis, N- and C-terminal sequence analysis, spectroscopic (UV, CD, ORD) analysis, peptide mapping, electrophoresis, chromatographic purity profile methods, potency/activity assays, etc. It must be emphasized that no one method is considered to be sufficient in itself, and that multiple methods are necessary to completely characterize and/or control such products.
For example, amino acid analysis for proteins with molecular weights above about 16 kilodaltons is known to be of very limited value. While useful for identification of the target protein, N-terminal and C-terminal sequencing only partially characterize the protein. Analysis of the primary structure, however, is insufficient to assure the biological potency of a protein, particularly since the potency depends on the protein conformation.
The conformation of proteins is usually analyzed by optical spectroscopy, such as UV spectroscopy, fluorescence spectroscopy, optical rotary dispersion, or circular dichroism. These methods are generally not sensitive enough to detect the subtle conformational changes caused by small alterations in the protein structure, especially if these changes do not affect side-chain chromophores from tryptophan, phenylalanine, tyrosine, and cysteine residues within the protein. Furthermore, these methods as well as others, such as electrophoresis, isoelectric focusing, differential scanning calorimetry, light scattering, ultracentrifugation, gel filtration, and immunological assays, only provide information about a particular structural or functional feature of a protein.
Chromatography is currently the most widely used analytical method for determining the purity of small organic drugs. Four modes of High-Performance Liquid Chromatography (HPLC) currently used for protein analysis are size-exclusion, ion-exchange, reversed-phase, and hydrophobic interaction chromatography. All these HPLC methods, though commonly employed to monitor the purity of biopharmaceuticals, are usually incapable of resolving proteins that differ by one or two residues or detecting other small changes in the macromolecular structure.
Hence, while chromatography is sufficient for determining whether a small organic drug is functional, the evaluation of a biopharmaceutical requires measurements of biological activity. Many of these measurements are the animal-based assays, particularly when the mechanism of action of the biopharmaceutical is not well defined. These assays are generally imprecise (with variability often 30% to 100%), time-consuming, and costly, and are not rugged. Cell culture assays can be used when the protein-based drug produces a measurable response in a cell-based system. The variability of these assays is much lower, often in the range of 10% to 30% or better (e.g., in vitro clot lysis assay has a variability of about 5%).
Physicochemical tests are much faster, more precise, and more reliable than biological assays. A physicochemical test providing information related to the biological potency of a protein-based drug would improve the control of the safety and efficacy of the drug.
Such a test should meet the following requirements: (1) it should provide information quantitatively related to the biological potency of a biopharmaceutical, (2) it should be capable of detecting minor changes in the structure of large macromolecules, (3) it should be especially sensitive to the structural changes affecting the efficacy of a biomacromolecule, (4) it should be sensitive to the presence of impurities in the product in quantities as small as 0.1 to 0.01 wt. %, (5) it should be simple, precise, and rugged, and (6) it should be time-, labor-, and cost-effective so as not to increase the overall cost of the product. Even if only some of these requirements were met, the test would improve the possibilities for assuring the safety and efficacy of biopharmaceuticals, such as recombinant human growth hormone (rhGH).
yet remain ignorant of those much brighter such as the fella with a Harvard degree in Medicine and subspecialty certification in endocrinology whom questioned the veracity of your "HCG" post, LMAO!
