Chemistry: analytical and immunological testing – Peptide – protein or amino acid – Glycoproteins
Patent
1998-01-26
2000-08-01
Wallenhorst, Maureen M.
Chemistry: analytical and immunological testing
Peptide, protein or amino acid
Glycoproteins
436 86, 436161, G01N 3368
Patent
active
06096555&
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a process for characterizing the glycosylation of glycoproteins and to an in-vitro process for determining the bioavailability of glycoproteins, which process is based on the "hypothetical charge number" (termed "N" below) and can be employed both for endogenous glycoproteins and exogenous glycoproteins.
In this connection, exogenous glycoproteins are, for example, recombinant therapeutic glycoproteins (such as interleukin 2, erythropoietin, tissue plasminogen activator or antithrombin III) which are obtained from mammalian cells. In recent years, these substances have aroused substantial interest in scientific, pharmaceutical and official institutions.
In this connection, endogenous glycoproteins are human or non-human (e.g. bovine) serum glycoproteins (such as human .alpha..sub.1 -acid glycoprotein, human transferrin or bovine fetuin, and also glycoproteins of other species such as chicken ovomucoid or porcine thyroglobulin).
The investigation, development and production of therapeutic glycoproteins, and their official and/or clinical licensing, requires elaborate analysis with regard to in-vivo half life, biological safety, product definition and batch consistency.
In this respect, the proportion of sialic acid (N-acetylneuraminic acid, Neu5Ac), in particular, has previously been an important parameter since it is assumed that the presence or absence of Neu5Ac has a decisive influence on the circulating half life of a glycoprotein in the blood and on its clearance. However, precise investigation of the carbohydrate side chains of a glycoprotein demands very elaborate and complex analysis which requires a high degree of expertise and a wide range of equipment (for example GC-MS, FAB-MS and high-resolution 1H NMR spectroscopy).
In particular, the authorities which are responsible for licensing therapeutic glycoproteins (e.g. EPO) still require, for safety reasons, the very elaborate, time-consuming, expensive but relatively inexact determination of the therapeutic activity of the glycoprotein in animal experiments (in-vivo assay). More advantageously, however, an in-vitro assay should be available which, firstly, can be carried out in a simple and reliable manner and, secondly, meets the justifiably high demands of the licensing authorities.
With regard to implementing additional determinations, some success has already been achieved in replacing the tedious and expensive methods of glycoanalysis with standardizable chromatographic methods (Hermentin et al. (1992) Anal. Biochem. 203, 281-289; idem., ibid., (1992) 206, 419-429). However, there has not previously been any parameter which would have satisfied the requirements of the licensing authorities as a replacement for an in-vivo assay for the bioavailability of a glycoprotein.
The present invention was therefore based on the technical problem of providing an in-vitro process which is suitable for determining the degree of glycosylation of a glycoprotein in such a simple and reliable manner that the process is suitable for replacing the known in-vivo processes, for example for determining bioavailability and batch consistency.
This technical problem is solved by providing the embodiments which are characterized in the patent claims.
It has been ascertained, surprisingly, that N correlates outstandingly well, and with good reproducibility, with the bioavailability/biological activity of a glycoprotein as found in in-vivo methods. Because of the good reproducibility and the analytical accuracy, N can advantageously also be employed in a process for determining batch consistency.
The present investigations allow the conclusion that N characterizes the "glycosylation status". Glycosylation can therefore be compared in a simple manner by determining N.
In connection with the present invention, the "glycosylation status" of a glycoprotein is understood as being the composition of the glycan pool comprising bi-, tri- and tetra-antennary glycans and their respective degree of sialylation (the content of bound N-acetylneuraminic
REFERENCES:
Aoyagi, "Carbohydrate-based Measurments on Alpha-fetaprotein in the Early Diagnosis of Heptocellular Carcinoma", GLYCO. J., 12(3):194-199 (Jun. 1995).
De Graaf et al., "Iflammation-induced Expresion of Sialyl Lewis X-containing Glycan Structures on .alpha..sub.1 -Acid Glycoprotein (Orosomucoid in Human Sera", J. Exp. Med., 177:657-666 (Mar. 993).
De Jong et al., "Transferrin Microheterogeneity as a Probe in Normal and Disease States", GLYCO. J., 12:219-266 (1995).
Hardy et al., "Monosaccharide Analysis of Glycoconjugates by Anion Exchange Chromotography with Pulsed Amperometric Detection", Analyt. Biochem., 170:54-62 (1988).
Hermentin et al., "The Mapping by High-pH Anion-Exchange Chromatography with Pulsed Amperometric Detection and Capilary Electrophoresis of the Carbohydrate Moieties of Human Plasma .alpha.-Acid Glycoprotein.sup.1,2 ", Analyt. Biochem., 206:419-429 (1992).
Hermentin and Seidat, "Micro-Scale Analysis of N-Acetylneuraminic Acid", GBF Monographs, 15:185-188 (1991).
Hermentin et al., "A Strategy for the Mapping of N-Glycans by High-pH Anion-Exchange Chromatography with Pulsed Amperometric Detection.sup.1,2 ", Analyt. Biochem., 203:281-289 (1992).
Hermentin et al., "The Hypothetical N-Glycan Charge: A Number That Characterizes Protein Glycosylation", Glycobiology, 6(2) 217-230 (1996).
Hokke et al., "Structural Analysis of the Sialylated--and O-linked Carbohydrate Chains of Recombinant Human Erythropoietin Expresses in Chinese Hamster Ovary Cells", Structural Determination Of Glycoprotein Glycans (Doctoral Thesis) 4:51-90 (1993).
Koch, "Einfuhrung in dies Biopharmazie", Osterreichische Apotheker-Zeitung, 29:313-320 (Apr. 1975).
Lee and Rice, "Fractionation of Glycopeptides and Oligosaccharides from Glycoproteins by HPLC", Glycobiology--A Practical Approach, ch. 3C, pp. 127-163 (1994).
Mackiewicz and Mackiewicz, "Glycoforms of Serum .alpha.1-Acid Glycoprotein as Markers of Inflammation and Cancer", GLYCO. J., 12:241-247 (1995).
Nimitz et al., "Structures of Sialylated Oligosaccharides of Human Erythropoietin Expressed in Recombinant BHK-21 Cells", Eur. J. Biochem., 213:39-56 (1993).
Nuck et al., "Optimized Deglycosylation Glycoproteins by Peptide-N.sup.4 --(N-acetyl-.beta.-glucosaminy)- asparagine Amidase from Flavobacterium meningosepticum", GLYCO. J., 7:279-286 (1990).
Orntoft and Bech, "Circulating Blood Group Related Carbohydrate Antigens as Tumour Markers", GLYCO. J., 12:200-205 (1995).
Ritschel, "Graphic Approach to Clinical Pharmacokinetics", 1 ed., pp. 1-18 (1984).
Shahangian et al., "Plasma Protein-Bound Sialic Acid in Patients with Colorectal Polyps of Known Histology", Clin. Chem., 37(2):200-204 (1991).
Turner et al., "Glycosylation of Alpha-1-Proteinase Inhibitor and Haptoglobin in Ovarian Cancer; Evidnece for Two Differenct Mechanisms", GLYCO. J., 12:211-218 (1995).
Watson et al., "Structure Determination of the Intact Major Sialylated Oligosaccharide Chains of Recombinant Human Erythropoietin Expressed in Chinese Hamster Ovary Cells", Glycobiology, 4(2):227-237 (1994).
Hermentin Peter
Witzel Reinhild
Dade Behring Marburg GmbH
Wallenhorst Maureen M.
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