1. Field of the Invention
The present invention is in the field of detection of drugs used for enhancing athletic performance and more specifically in the field of detection of use of natural peptide hormones produced by the techniques of genetic engineering.
2. Background and Description of Related Art
Performance Enhancement
Since the first athletic competition participants have made every attempt to improve and enhance their performance. To the present day this has been done by training, practice, nutrition and striving to maintain good health. Athletes have continued to improve, but there is always the temptation to take “shortcuts,” that is, to use various drugs to enhance athletic training and performance. The use of “steroids” by weight lifters and bodybuilders is well known. Similar drugs have also become popular with many other athletes as an aid to training and performance enhancement. Fortunately, many of these compounds are relatively easy to detect in an athlete's blood or other body fluids, thus making steroid use much less attractive. Even difficult to detect steroids are subject to detection by analysis of carbon isotope ratios.
Endurance athletes have been interested in increasing their stamina by “blood doping”. Originally, this practice was carried out by collecting and storing the athlete's own blood followed by transfusing the athlete with a concentrate of his or her own red blood cells immediately prior to a competition. This significantly enhances athletic performance by increasing the blood's ability to carry oxygen to the muscles. The downside is that transfusions are cumbersome and require significant medical participation, thereby increasing the chance of detection. Also, the results are rather transitory as the body eliminates the excess red blood cells. Further, the practice is dangerous in that it increases the viscosity of the blood and can lead to cardiovascular disease.
Erythropoietin, a glycoprotein hormone produced primarily the kidney and to a lesser extent in the liver. It is one of at least 20 polypeptide factors that control blood cell formation (1), stimulates the production of red blood cells by bone marrow and can produce the effects of “blood doping” with no need for painful transfusions. All that is required is a tiny injection of EPO1 every few days. However, until recently EPO had to be purified from human urine and was, hence, costly and virtually unavailable. However, availability of recombinant proteins produced for therapeutic medical purposes has become a reality in recent years. Although there are only a handful of recombinant proteins/drugs on the market presently, there are over 100 such products at various stages of the FDA approval process. Unfortunately, the current commercial availability of EPO produced by recombinant DNA techniques has provided an easy means for the athlete who wishes to participate in “blood doping”. In fact, a U.S. patent has even been granted on a process to increase the hematocrit of a normal mammal through the use of EPO (U.S. Pat. No. 5,541,158). Furthermore, it is quite possible that recombinant products (e.g., the various human growth hormones) other than EPO will also have the potential for abuse by athletes so that the methods disclosed herein are applicable to situations other than EPO administration. 1Abbreviations: BHK: baby hamster kidney, CHO: Chinese hamster ovary, EPO: erythropoietin, uHuEPO: urinary human EPO, rHuEPO: recombinant human EPO, LacNAc: N-acetyllactosamine; HPAEC-PAD: high pH anion-exchange chromatography with pulsed amperometric detection; ELISA: enzyme linked immunoassay.
There are presently no reliable methods available for detecting the abuse of EPO by athletes. There is sufficient indication in the sports medicine literature to strongly suggest that athletes may, in fact, be abusing EPO (2–4). A great deal of interest in the swimming community has resulted from a strong suggestion that the Chinese women swimmers may be using a number of performance enhancing substances including EPO (9). A number of investigators have measured EPO levels in trained athletes, untrained controls, before and after competition in trained athletes (5–8) with varied results. It has been suggested that some of the variability may be due to problems with the bioassay for EPO activity as well as individual variability in the population. This could pose a significant problem in detecting artificially administered EPO because it is likely to be impossible to prove that a high level of EPO is a result of “blood doping” as opposed to a naturally high level. In fact, individuals with “athletic ability” might inherently have a higher endogenous EPO level.
Erythropoietin:
The structure and function of the carbohydrate side chains of Erythropoietin have been the subject of extensive investigation (10–17). Erythropoietin is known to have three N-linked oligosaccharide chains [at asparagine 24, 38, and 83] and one O-linked oligosaccharide chain per molecule [at serine 126]. The carbohydrate side chains make up almost 40% of the total mass of the molecule. At least 83 different sugar chain structures have been determined for the asparagine-linked side chains of human erythropoietins (29) and at least four different sugar chain structures have been determined for the O-linked side chains on serine 126. The structures of the N-linked sugar chains of rHuEPO have been well characterized. However, the structures of the mucin-type chains of uHuEPO have not been as well characterized as the N-linked chains.
Differences in the molar ratio (%) of ten different N-linked side chains were summarized in a review by Takeuchi and Kobata (10). They presented data for a biantennary, four triantennary, two tetraantennary side chains and three tetraantennary side chains with 1, 2, and 3 LacNAc repeating units. Past work has demonstrated differences in the oligosaccharide chains of the recombinant EPO and endogenous EPO (11–18). In addition to the carbohydrate differences discussed above, there are also differences in the sialic acid linkages due to the deficiency of a specific sialyltransferase in CHO cells which are used for the expression of the recombinant protein (15). ARANESP™ (trademark of Amgen, Inc. for the generic peptide Darbepoitin alfa) is a modified form of recombinant EPO which has two additional N-linked carbohydrate side chains. Specifically, two amino acid substitutions have been introduced at Ala 30 and a Trp 88 to produce a form of EPO which has a total of 5 N-linked carbohydrate side chains and one O-linked carbohydrate side chain. Since the molecule is produced in CHO cells as EPO, the additional carbohydrate side chains have the same carbohydrate structures and they still have the same heterogeneity as the original carbohydrate side chains in recombinant EPO (30). Therefore the work presented here on EPO is directly applicable to ARANESP™ (Darbepoitin alfa).
Principles Behind Present Invention:
The present invention is possible because the DNA for a protein only codes for the primary amino acid sequence of that protein. If such a protein is a glycoprotein, then one can state a priori that the DNA for the protein does not code for the carbohydrate side chains of that glycoprotein although the peptide sequence may determine the sites of glycosylation. Thus, the glycosylation of a recombinant gene product is dependent on the glycosylation machinery of the cell line in which the gene is expressed. In essence, glycosylation is dependent on the array of glycosyltransferases and pools of sugar nucleotide precursors available in the expressing cells. It has been well documented that, in the case of recombinant glycoproteins, the nature of the carbohydrate side chains is dependent on the cell line in which the glycoprotein is produced and variables in the culture conditions(19, 20). Since it is virtually impossible that tissue culture and human cells in an intact individual would have identical transferases and precursor pools, a given glycosylation pattern can potentially act as a fingerprint for the source of a glycoprotein. The heterogeneity of glycoproteins that is the result of variations the carbohydrate moieties of glycoproteins has given rise to the term glycoforms to describe the multiple forms of the same glycoprotein (i.e., the peptide sequence is the same).
Thus, one or more glycosylation sites on a recombinant peptide may have carbohydrate chains that are not present on the endogenous glycopeptide/glycoprotein. Although sugar groups are often considered “nonantigenic,” antibodies can in fact be produced under some conditions. For example, the glycopeptides can be conjugated to bovine serum albumin or Keyhole Limpet Hemocyanin (KLH) to increase their antigenicity. Either polyclonal and monoclonal antibodies to the isolated glycopeptide can then be produced. The resulting library of antibodies can then be screened for ability to recognize the desired glycopeptide. Next the antibodies that recognized the glycopeptide can be screened for those that recognize the intact recombinant molecule. Finally, those antibodies that recognize the desired glycosylated site on the intact recombinant molecule can be tested to determine if they recognize the endogenous glycoprotein molecule, in the case of EPO uHuEPO. At that point, the selection process identifies antibodies that recognize the carbohydrate chain that is unique to the recombinant molecule but that do not recognize the endogenous molecule. These antibodies can then be used to make an enzyme-linked immunoassay kit (ELISA) which recognizes only administered recombinant EPO and ignores endogenous, natural EPO or any other antibody-based detection method can be used.