Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-11-17
2002-10-29
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S005000, C435S006120, C435S325000, C435S348000, C435S320100
Reexamination Certificate
active
06472175
ABSTRACT:
FIELD OF THE INVENTION
The invention pertains to the field of protein expression systems. More particularly, the invention pertains to optimizing the N-linked glycosylation of proteins in a baculovirus expression system.
BACKGROUND OF THE INVENTION
Glycobiology is a newly emerging area of biotechnology. Most of the extracellular proteins of higher animals are glycoproteins, including proteins of pharmaceutical interest such as erythropoietin, tissue plasminogen, interleukins and interferons. The ubiquity and diversity of glycoproteins is matched by the breadth of functions that they have in a wide range of important biological processes. For instance, glycosylation plays an important role in hormone signal transduction and in the biological activity of immunoglobulins. Glycoproteins also play a structural role in connective tissues such as collagen. Glycosylation of proteins clearly represents one of the most important co- and post-translational events.
Glycoproteins are composed of a polypeptide chain covalently bound to one or more carbohydrate moieties. There are two broad categories of glycoproteins with carbohydrates coupled through either N-glycosidic or O-glycosidic linkages to their constituent protein. The N- and O-linked glycans are attached to polypeptides through asparagine-N-acetyl-D-glucosamine and serine (threonine)-N-acetyl-D-galactosamine linkages, respectively. Complex N-linked oligosaccharides do not contain terminal mannose residues. They contain only terminal N-acetylglucosamine, galactose, and/or sialic acid residues. Hybrid oligosaccharides contain terminal mannose residues as well as terminal N-acetylglucosamine, galactose, and/or sialic acid residues.
With N-linked glycoproteins, an oligosaccharide precursor is attached to the amino group of asparagine during peptide synthesis in the endoplasmic reticulum. The oligosaccharide moiety is then sequentially processed by a series of specific enzymes that delete and add sugar moieties. The processing occurs in the endoplasmic reticulum and continues with passage through the cis-, medial- and trans-Golgi apparatus (FIGS.
1
A and B).
The regulation of the glycosylation process is complex because it contains both synthetic and degradative steps that are controlled by very specific enzymes. Currently, the regulation of glycoprotein synthesis and processing is not well understood.
Glycosylation in the Baculovirus Expression System
It has been estimated that the baculovirus-polyhedrin protein can constitute up to 50% of the total protein mass at cell death. The polyhedrin gene is one of the most highly expressed viral genes described. One of the reasons for this high expression level is that the polyhedrin gene is under the transcriptional control of a very strong promoter. Replacement of the polyhedrin gene open-reading-frame (ORF) with the ORF of a foreign gene under the control of the polyhedrin gene promoter results in high levels of expression of the foreign gene product. Production levels as high as 1 mg/10
6
cells have been obtained. This method of producing foreign proteins is referred to as the baculovirus expression vector system (BEVS).
Hundreds of proteins have been expressed in stationary insect cell cultures with the baculovirus expression vector system (BEVS). There is substantial pharmaceutical interest in using the BEVS to produce commercial products in insect cells. The BEVS has several advantages as a recombinant protein production system, such as 4-6 weeks from gene isolation to BEVS expression, high production levels and the absence of adventitious viruses (commonly found in mammalian tissue culture cells). Equally important is the fact that insect cells are able to recognize the co- and post-translational signals of higher eukaryotes, resulting in processing such as phosphorylation, proteolytic processing, carboxyl methylation, and glycosylation. Of all these co- and post-translational processing events, glycosylation has been found to have the greatest influence on many of the physical and functional properties of proteins.
Altering the type of glycan modifying a glycoprotein can have dramatic affects on a protein's antigenicity, structural folding, solubility, and in vivo bioactivity and stability. Also, varying the number and composition of the oligosaccharide moieties can significantly alter the physical characteristics for many glycoproteins. In particular, it has been demonstrated that terminal sialic acid residues play an extremely important role in defining the in vivo biological activity of many glycoproteins. For example, terminal sialic acid residues have been demonstrated to be very important in defining the immunogenicity of glycoproteins.
The absence of sialic acid has been found to influence the biological activity of many proteins. In particular, the specific activities of proteins, such as tissue plasminogen (used clinically to dissolve blood clots) and erythropoietin (which stimulates maturation of red blood cells), have been found to be dramatically altered by the removal of terminal sialic acid residues. Furthermore, the specific recognition of oligosaccharide moieties is the primary mechanism for protein clearance from the circulatory system. Therefore, differences in the oligosaccharide structure, particularly the presence or absence of sialic acid, can significantly affect both the in vivo and in vitro properties of glycoproteins. Thus, if insect cells are used to produce therapeutic glycoproteins, it is critical to generate glycoproteins with terminal sialic acid residues.
Experience with the expression of N-linked glycoproteins using the BEVS clearly indicates that insect cells generally recognize the same signals for glycosylation sites as mammalian cells. The N-linked glycosylation pathway is outlined in
FIGS. 1A and B
. Glycosylation begins with the attachment of the dolichol-phosphate precursor oligosaccharide. Following this initial step, there is efficient removal of glucose residues by &agr;-glucosidase I and II and subsequent removal of mannose residues with endoplasmic reticulum mannosidase and Golgi mannosidase I. This glycan trimming process appears to progress in a proficient fashion in lepidopteran larvae and tissue culture cells.
Following these trimming events, mammalian glycan processing is typically subject to the sequential enzymatic addition of N-acetylglucosamine (GIcNAc), sometimes fucose, followed by galactose (Gal) and sialic acid residues (FIGS.
1
A and B). However, mammalian glycoproteins that normally have complex glycans with terminal sialic acid residues when they are produced in mammalian cells, are expressed in the BEVS in insect cells with oligosaccharides containing high mannose (Man
8-5
GlcNAc
2
) or paucimannose (Man
2-3
GlcNAc
2
) structures (note the absence of sialic acid residues). Some of the structures contain &agr;1,6 linked fucose and/or terminal GlcNAc residues. Note that in the foregoing descriptions of oligosaccharide structures, the term “Man” is an abbreviation used to described mannose.
Early studies of N-linked glycoproteins expressed in the BEVS suggested that insect cells were not able to add GlcNAc, Gal or sialic acid residues (Wathen et al., 1989; Kuroda et al., 1990; Kretzschmar et al., 1994). However, the enzymes required for the addition of GlcNAc and Gal residues have been identified in insect cell lines derived from
B. mori
(Bm-N),
Mamestra brassicae
(IZD-Mb-05030, referred to as Mb) and
Spodoptera frugiperda
(IPLB-SF21AE, referred to as Sf-9 and Sf-21). &bgr;1,2-N-acetylglucosaminyltransferase I (GlcNAc-T-I) activity has been found in Bm-N, Mb, Sf-9 and Sf-21 tissue culture cells (Altmann el al., 1993). However, it should be noted that the high level of GIcNAc-T-I activity found in the Bm-N, Mb and Sf-21 tissue culture cells by Altmann et al. (1993) was not reflected in the N-linked oligosaccharide structures associated with cell membranes characterized by Kubelka et al. (1994). Only a small percentage of the membrane-associated structures had terminal GlcNAc residues.
Following the addition of
Boyce Thompson Institute For Plant Research, Inc.
Brown & Michaels PC
Guzo David
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