Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Rodent cell – per se
Reexamination Certificate
2000-04-21
2003-01-14
Low, Christopher S. F. (Department: 1653)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Rodent cell, per se
C435S069600, C435S358000, C435S252300, C530S395000
Reexamination Certificate
active
06506598
ABSTRACT:
BACKGROUND OF THE INVENTION
1 Field of the Invention
The present invention concerns a process for the production of glycoproteins in mammalian cell culture. More specifically, the invention provides a process for producing glycoproteins in mammalian cells that results in enhanced occupancy of an N-linked glycosylation site occupied only in a fraction of a glycoprotein. A process for increasing the fraction of Type I tissue plasminogen activator (t-PA) in a mammalian cell culture is specifically disclosed.
2. Description of Related Disclosures and Technology
Glycoproteins
Glycoproteins, many of which have been produced by techniques of recombinant DNA technology, are of great importance as diagnostic and therapeutic agents. In a eukaryotic cell environment, glycosylation is attached to a secreted or membrane-spanning protein by co- and post-translational modification. Proteins destined for the cell surface are first co-translationally translocated into the lumen of the endoplasmic reticulum (ER) mediated by a signal sequence at or near the amino terminus of the nascent chain. Inside the ER, the signal sequence is usually removed and a high-mannose core oligosaccharide unit is attached to the asparagine (N) residue(s) present as part of the sequence Asn-X-Ser/Thr, where X is any amino acid except, perhaps, proline.
The efficiency of this co-translational glycosylation step is dependent on the presentation of an appropriate conformation of the peptide chain as it enters the endoplasmic reticulum (Imperiali and O'Connor,
Pure & Applied Chem.,
70: 33-40 (1998)). Potential N-linked glycosylation sites may no longer be accessible after the protein has folded (Komfeld & Komfeld,
Ann Rev. Biochem.
54:631-664 (1985)). Proteins next move from the ER to the Golgi apparatus where further modifications, such as sulfation and processing of the high-mannose oligosaccharide chain to a complex-type oligosaccharide, occur and the proteins are directed to their proper destinations.
N-linked oligosaccharides can have a profound impact on the pharmaceutical properties of glycoprotein therapeutics (e.g., in vivo half-life and bioactivity). Different bioprocess parameters (e.g., bioreactor type, pH, media composition, and ammonia) have been shown to affect protein glycosylation significantly. Changes in terminal glycosylation (sialylation and galactosylation) and N-glycan branching are the most frequently observed alterations.
The Carbohydrate Structure of Tissue Plasminogen Activator
Tissue plasminogen activator (t-PA), a glycoprotein, is a multidomain serine protease whose physiological role is to convert plasminogen to plasmin, and thus to initiate or accelerate the process of fibrinolysis. Initial clinical interest in t-PA was raised because of its relatively high activity in the presence, as compared to the absence, of fibrin. Wild-type t-PA is a poor enzyme in the absence of fibrin, but the presence of fibrin strikingly enhances its ability to activate plasminogen. Recombinant human t-PA is used therapeutically as a fibrinolytic agent in the treatment of acute myocardial infarction and pulmonary embolism, both conditions usually resulting from an obstruction of a blood vessel by a fibrin-containing thrombus.
In addition to its striking fibrin specificity, t-PA exhibits several further distinguishing characteristics:
(a) T-PA differs from most serine proteases in that the single-chain form of the molecule has appreciable enzymatic activity. Toward some small substrates, and toward plasminogen in the absence of fibrin, two-chain t-PA has greater activity than one-chain t-PA. In the presence of fibrin, however, the two forms of t-PA are equally active (Rijken et al.,
J. Biol. Chem.
257: 2920-2925 (1982); Lijnen et al.,
Thromb Haemost.,
64: 61-68 (1990); Bennett et al.,
J. Biol. Chem.,
266: 5191-5201 (1991)). Most other serine proteases exist as zymogens and require proteolytic cleavage to a two-chain form to release full enzymatic activity.
(b) The action of t-PA in vivo and in vitro can be inhibited by a serpin, PAI-1 (Vaughan et al.,
J. Clin. Invest.,
84: 586-591 (1989); Wiman et al.,
J. Biol. Chem.,
259: 3644-3647 (1984)).
(c) T-PA binds to fibrin in vitro with a K
d
in the &mgr;M range.
(d) T-PA has a rapid in vivo clearance that is mediated by one or more receptors in the liver (Nilsson et al.,
Thromb. Res.,
39: 511-521 (1985); Bugelski et al.,
Throm. Res.,
53: 287-303 (1989); Morton et al.,
J. Biol. Chem.,
264: 7228-7235 (1989)).
A substantially pure form of t-PA was first produced from a natural source and tested for in vivo activity by Collen et al., U.S. Pat. No. 4,752,603 issued Jun. 21, 1988 (see also Rijken et al.,
J. Biol. Chem.,
256: 7035 (1981)). Pennica et al. (
Nature,
301: 214(1983)) determined the DNA sequence of t-PA and deduced the amino acid sequence from this DNA sequence (U.S. Pat. No. 4,766,075 issued Aug. 23, 1988).
Human wild-type t-PA has potential N-linked glycosylation sites at amino acid positions 117,184,218, and 448. Recombinant human t-PA (ACTIVASE® t-PA) produced by expression in CHO cells was reported to contain approximately 7% by weight of carbohydrate, consisting of a high-mannose oligosaccharide at position 117, and complex oligosaccharides at Asn-184 and Asn-448 (Vehar et al., “Characterization Studies of Human Tissue Plasminogen Activator produced by Recombinant DNA Technology, ”
Cold Spring Harbor Symposia on Quantitative Biology,
LI:551-562 (1986)).
Position 218 has not been found to be glycosylated in native t-PA or recombinant wild-type t-PA. Sites 117 and 448 appear always to be glycosylated, while site 184 is thought to be glycosylated only in a fraction of the molecules. The t-PA molecules that are glycosylated at position 184 are termed Type I t-PA, and the molecules that are not glycosylated at position 184 are termed Type II t-PA. In melanoma-derived t-PA, the ratio of Type I to Type II t-PA is about 1:1. The most comprehensive analysis of the carbohydrate structures of CHO cell-derived human t-PA was carried out by Spellman et al.,
J. Biol. Chem.
264: 14100-14111 (1989), who showed that at least 17 different Asn-linked carbohydrate structures could be detected on the protein. These ranged from the high-mannose structures at position 117 to di-, tri-, and tetra-antennary N-acetyllactosamine-type structures at positions 184 and 448. Type I and Type II t-PAs were reported to be N-glycosylated in an identical way at Asn-117 and Asn-448 positions, when isolated from the same cell line. For further details, see also Parekh et al.,
Biochemistry,
28: 7644-7662 (1989). The specific fibrinolytic activity of Type II t-PA has been shown to be about 50% greater than that of Type I t-PA (Einarsson et al.,
Biochim. Biophys. Acta,
830: 1-10 (1985)). Further, increased Type I is correlated with increased half-life (Cole et al.,
Fibrinolysis,
7: 15-22 (1993)). However, Type II t-PA, which lacks a portion of carbohydrate associated with Type I t-PA, as well as desialated t-PA, demonstrated a longer T½ beta than standard t-PA (Beebe and Aronson,
Thromb. Res.
51: 11-22 (1988)).
Analysis of the sequence of t-PA has identified the molecule as having five domains. Each domain has been defined with reference to homologous structural or functional regions in other proteins such as trypsin, chymotrypsin, plasminogen, prothrombin, fibronectin, and epidermal growth factor (EGF). These domains have been designated, starting at the N-terminus of the amino acid sequence of t-PA, as the finger (F) domain from amino acid 1 to about amino acid 44, the growth factor (G) domain from about amino acid 45 to about amino acid 91 (based on homology with EGF), the kringle-1 (K1) domain from about amino acid 92 to about amino acid 173, the kringle-2 (K2) domain from about amino acid 180 to about amino acid 261, and the serine protease (P) domain from about amino acid 264 to the carboxyl terminus at amino acid 527. These domains are situated essentially adjacent to each other, and are connected by short “linker” regions. These linker regions
Andersen Dana C.
Bridges Tiffany M.
Gawlitzek Martin
Hoy Cynthia A.
Genentech Inc.
Hasak Janet E.
Low Christopher S. F.
Schnizer Holly
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