U.S. patent application number 10/007331 was filed with the patent office on 2002-08-29 for practical in vitro sialylation of recombinant glycoproteins.
This patent application is currently assigned to Cytel Corporation. Invention is credited to Bayer, Robert J., Paulson, James C., Sjoberg, Eric.
Application Number | 20020119516 10/007331 |
Document ID | / |
Family ID | 21884346 |
Filed Date | 2002-08-29 |
United States Patent
Application |
20020119516 |
Kind Code |
A1 |
Paulson, James C. ; et
al. |
August 29, 2002 |
Practical in vitro sialylation of recombinant glycoproteins
Abstract
This invention provides methods for practical in vitro
sialylation of glycoproteins, including recombinantly produced
glycoproteins. The methods are useful for large-scale modification
of sialylation patterns.
Inventors: |
Paulson, James C.; (Del Mar,
CA) ; Bayer, Robert J.; (San Diego, CA) ;
Sjoberg, Eric; (San Diego, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Cytel Corporation
|
Family ID: |
21884346 |
Appl. No.: |
10/007331 |
Filed: |
November 9, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10007331 |
Nov 9, 2001 |
|
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09007741 |
Jan 15, 1998 |
|
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60035710 |
Jan 16, 1997 |
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Current U.S.
Class: |
435/68.1 ;
435/193 |
Current CPC
Class: |
C12N 9/1048 20130101;
C12P 21/005 20130101 |
Class at
Publication: |
435/68.1 ;
435/193 |
International
Class: |
C12P 021/06; C12N
009/10 |
Claims
What is claimed is:
1. A method of sialylating a saccharide group on a recombinant
glycoprotein, the method comprising contacting a saccharide group
which comprises a galactose or N-acetylgalactosamine acceptor
moiety on a recombinant glycoprotein with a sialic acid donor
moiety and a recombinant sialyltransferase in a reaction mixture
which provides reactants required for sialyltransferase activity
for a sufficient time and under appropriate conditions to transfer
sialic acid from said sialic acid donor moiety to said saccharide
group.
2. The method of claim 1, wherein the sialic acid donor moiety is
CMP-sialic acid.
3. The method of claim 2, wherein the CMP-sialic acid is
enzymatically generated in situ.
4. The method of claim 1, wherein the sialyltransferase is a
recombinant eukaryotic sialyltransferase which substantially lacks
a membrane-spanning domain.
5. The method of claim 1, wherein the sialyltransferase includes a
sialyl motif which has an amino acid sequence that is at least
about 40% identical to a sialyl motif from a sialyltransferase
selected from the group consisting of ST3Gal I, ST6Gal I, and
ST3Gal III.
6. The method of claim 1, wherein the sialyltransferase is a
recombinant ST3Gal III.
7. The method of claim 6, wherein the sialyltransferase is a
recombinant rat ST3Gal III.
8. The method of claim 1, wherein the sialyltransferase is a
recombinant ST3Gal IV.
9. The method of claim 1, wherein the sialyltransferase is a
recombinant ST6Gal I.
10. The method of claim 1, wherein the sialyltransferase is a
recombinant ST3Gal I.
11. The method of claim 10, wherein the reaction mixture comprises
a second recombinant sialyltransferase, which second recombinant
sialyltransferase is an ST3Gal III.
12. The method of claim 1, wherein the sialyltransferase is a
recombinant bacterial sialyltransferase.
13. The method of claim 12, wherein the bacterial sialyltransferase
has an amino acid sequence which is at least 50% identical to an
amino acid sequence of a Neisseria meningitidis
2,3-sialyltransferase.
14. The method of claim 13, wherein the bacterial sialyltransferase
is a Neisseria meningitidis 2,3-sialyltransferase.
15. The method of claim 12, wherein the bacterial sialyltransferase
has an amino acid sequence which is at least 50% identical to an
amino acid sequence of a Photobacterium damsela
2,6-sialyltransferase.
16. The method of claim 15, wherein the bacterial sialyltransferase
is a Photobacterium damsela 2,6-sialyltransferase.
17. The method of claim 12, wherein the bacterial sialyltransferase
has an amino acid sequence which is at least 50% identical to an
amino acid sequence of a Haemophilus 2,3-sialyltransferase.
18. The method of claim 17, wherein the sialyltransferase is a
Haemophilus 2,3-sialyltransferase.
19. The method of claim 12, wherein the bacterial sialyltransferase
has an amino acid sequence which is at least 50% identical to an
amino acid sequence of a Campylobacter jejuni
2,3-sialyltransferase.
20. The method of claim 19, wherein the sialyltransferase is a
Campylobacter jejuni 2,3-sialyltransferase.
21. The method of claim 1, wherein the sialyltransferase is
produced by recombinant expression of a sialyltransferase in a host
cell selected from the group consisting of an insect cell, a
mammalian cell, and a fungal cell.
22. The method of claim 21, wherein the host cell is an Aspergillus
niger cell.
23. A method of sialylating a saccharide group on a recombinant
glycoprotein, the method comprising contacting a saccharide group
which comprises a galactose or an N-acetylgalactosamine acceptor
moiety on a recombinant glycoprotein with a sialic acid donor
moiety and a bacterial sialyltransferase in a reaction mixture
which provides reactants required for sialyltransferase activity
for a sufficient time and under appropriate conditions to transfer
sialic acid from said sialic acid donor moiety to said saccharide
group.
24. The method of claim 23, wherein the bacterial sialyltransferase
has an amino acid sequence which is at least 50% identical to an
amino acid sequence of a Photobacterium damsela
2,6-sialyltransferase.
25. The method of claim 24, wherein the bacterial sialyltransferase
is a Photobacterium damsela 2,6-sialyltransferase.
26. The method of claim 23, wherein the bacterial sialyltransferase
has an amino acid sequence which is at least 50% identical to an
amino acid sequence of a Neisseria meningitidis
2,3-sialyltransferase.
27. The method of claim 26, wherein the sialyltransferase is a
Neisseria meningitidis 2,3-sialyltransferase.
28. The method of claim 23, wherein the bacterial sialyltransferase
has an amino acid sequence which is at least 50% identical to an
amino acid sequence of a Campylobacter jejuni
2,3-sialyltransferase.
29. The method of claim 28, wherein the sialyltransferase is a
Campylobacter jejuni 2,3-sialyltransferase.
30. The method of claim 23, wherein the bacterial sialyltransferase
has an amino acid sequence which is at least 50% identical to an
amino acid sequence of a Haemophilus 2,3-sialyltransferase.
31. The method of claim 30, wherein the sialyltransferase is a
Haemophilus 2,3-sialyltransferase.
32. A method for in vitro sialylation of saccharide groups present
on a glycoprotein, said method comprising contacting said
saccharide groups with a sialyltransferase, a sialic acid donor
moiety, and other reactants required for sialyltransferase activity
for a sufficient time and under appropriate conditions to transfer
sialic acid from said sialic acid donor moiety to said saccharide
group, wherein said sialyltransferase is present at a concentration
about 50 mU per mg of glycoprotein or less.
33. The method of claim 32, wherein the sialyltransferase is
present at a concentration of between about 5-25 mU per mg of
glycoprotein.
34. The method of claim 32, wherein the sialyltransferase is
present at a concentration of between about 10-50 mU/ml of reaction
mixture and the glycoprotein is present in the reaction mixture at
a concentration of at least about 2 mg/ml.
35. The method of claim 32, wherein the method yields a
glycoprotein having sialylation of at least about 80% of terminal
galactose residues present on the saccharide groups.
36. The method of claim 32, wherein the sialyltransferase is a
recombinant sialyltransferase.
37. The method of claim 36, wherein the sialyltransferase
substantially lacks a membrane-spanning domain.
38. The method of claim 32, wherein the sialyltransferase includes
a sialyl motif which has an amino acid sequence that is at least
about 40% identical to a sialyl motif from a sialyltransferase
selected from the group consisting of ST3Gal I, ST6Gal I, and
ST3Gal III.
39. The method of claim 32, wherein the sialyltransferase is an
ST3Gal III.
40. The method of claim 39, wherein the ST3Gal III is a rat ST3Gal
III.
41. The method of claim 32, wherein the sialyltransferase is an
ST3Gal IV.
42. The method of claim 32, wherein the sialyltransferase is an
ST3Gal I.
43. The method of claim 42, wherein the reaction mixture comprises
a second recombinant sialyltransferase, which second recombinant
sialyltransferase is an ST3Gal III.
44. The method of claim 32, wherein the sialyltransferase is a
bacterial sialyltransferase.
45. The method of claim 44, wherein the bacterial sialyltransferase
is a recombinant sialyltransferase.
46. The method of claim 44, wherein the bacterial sialyltransferase
has an amino acid sequence which is at least 50% identical to an
amino acid sequence of a Neisseria meningitidis
2,3-sialyltransferase.
47. The method of claim 46, wherein the bacterial sialyltransferase
is a Neisseria meningitidis 2,3-sialyltransferase.
48. The method of claim 44, wherein the bacterial sialyltransferase
has an amino acid sequence which is at least 50% identical to an
amino acid sequence of a Photobacterium damsela
2,6-sialyltransferase.
49. The method of claim 48, wherein the bacterial sialyltransferase
is a Photobacterium damsela 2,6-sialyltransferase.
50. The method of claim 44, wherein the bacterial sialyltransferase
has an amino acid sequence which is at least 50% identical to an
amino acid sequence of a Campylobacter jejuni
2,3-sialyltransferase.
51. The method of claim 50, wherein the sialyltransferase is a
Campylobacter jejuni 2,3-sialyltransferase.
52. The method of claim 44, wherein the bacterial sialyltransferase
has an amino acid sequence which is at least 50% identical to an
amino acid sequence of a Haemophilus 2,3-sialyltransferase.
53. The method of claim 52, wherein the sialyltransferase is a
Haemophilus 2,3-sialyltransferase.
54. The method of claim 32, wherein the sialic acid donor moiety is
CMP-sialic acid.
55. The method of claim 54, wherein the CMP-sialic acid is
enzymatically generated in situ.
56. The method of claim 32, wherein the sialic acid is selected
from the group consisting of NeuAc and NeuGc.
57. A method for in vitro sialylation of saccharide groups present
on a glycoprotein, the method comprising contacting the saccharide
groups with an ST3Gal III sialyltransferase, a sialic acid donor
moiety, and other reactants required for sialyltransferase activity
for a sufficient time and under conditions to transfer sialic acid
from said sialic acid donor moiety to said saccharide group,
wherein said ST3Gal III sialyltransferase is present at a
concentration of less than about 50 mU per mg of glycoprotein.
58. The method of claim 57, wherein the method further comprises
contacting the saccharide groups with an ST6GalI sialyltransferase.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 60/035,710, filed Jan. 16, 1997, which is
incorporated herein by reference in its entirety for all
purposes.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention pertains to the field of in vitro sialylation
of glycoproteins, including recombinant glycoproteins.
[0004] 2. Background
[0005] The circulatory lifetime of glycoproteins in the blood is
highly dependent on the composition and structure of its N-linked
carbohydrate groups. This fact is of direct relevance for
therapeutic glycoproteins which are intended to be administered
parenterally. In general, maximal circulatory half life of a
glycoprotein requires that its N-linked carbohydrate groups
terminate in the sequence NeuAc-Gal-GlcNAc. Without the terminal
sialic acid (NeuAc), the glycoprotein is rapidly cleared from the
blood by a mechanism involving the recognition of the underlying
N-acetylgalactosamine (GalNAc) or galactose (Gal) residues (Goochee
et al. (1991) Bio/Technology 9: 1347-1355). For this reason,
ensuring the presence of terminal sialic acid on N-linked
carbohydrate groups of therapeutic glycoproteins is an important
consideration for their commercial development.
[0006] In principle, mammalian cell culture systems used for
production of most therapeutic glycoproteins have the capacity to
produce glycoproteins with fully sialylated N-linked carbohydrate
groups. In practice, however, optimal glycosylation is often
difficult to achieve. Under the conditions of large scale
production, overproduction of the glycoprotein by the cell can
outstrip its ability to keep up with glycosylation, and this
capability can be positively and negatively influenced by many
subtle variables in culture conditions (Goochee et al.,
supra.).
[0007] Production of glycoproteins in transgenic animals has some
of the same problems as mammalian cell culture. While the
"production" of a glycoprotein is inherently better controlled, it
is also less susceptible to manipulation. If glycosylation is not
complete, there is little that can be done with the animals to
alter the outcome. With transgenic animals there is often another
problem. While the predominant sialic acid in humans is
N-acetyl-neuraminic acid (NeuAc), goats, sheep and cows all produce
a large fraction of their total sialic acid as
N-glycolyl-neuraminic acid (NeuGc). Although impact of this
modification is not yet fully explored from a functional or
regulatory perspective, it is known that the NeuGc substitution is
antigenic in humans (Varki (1992) Glycobiology 2: 25-40).
[0008] Since the most important problems associated with
glycosylation of commercially important recombinant and transgenic
glycoproteins, involve terminal sialic acid, a need exists for an
in vitro procedure to enzymatically "cap" carbohydrate chains that
lack a terminal sialic acid. With such a procedure, the problem
encountered with transgenic glycoproteins could also be addressed
by resialylation with NeuAc once the "non-human" sialic acid NeuGc
was removed. The ideal method would employ a sialyltransferase that
is capable of efficiently sialylating N-linked or O-linked
oligosaccharides of recombinant glycoproteins on a practical scale.
The present invention fulfills these and other needs.
SUMMARY OF THE INVENTION
[0009] The present invention provides methods for in vitro
sialylation of saccharide groups present on a recombinantly
produced glycoprotein. The methods comprise contacting the
saccharide groups with a sialyltransferase, a sialic acid donor
moiety, and other reactants required for sialyltransferase activity
for a sufficient time and under appropriate conditions to transfer
sialic acid from the sialic acid donor moiety to said saccharide
group.
[0010] In a preferred embodiment, the methods are carried out using
sialyltransferase at a concentration of about 50 mU per mg of
glycoprotein or less, preferably between about 5-25 mU per mg of
glycoprotein. Typically, the concentration of sialyltransferase in
the reaction mixture will be between about 10-50 mU/ml, with the
glycoprotein concentration being at least about 2 mg/ml of reaction
mixture. In a preferred embodiment, the method results in
sialylation of greater than about 80% of terminal galactose
residues present on said saccharide groups. Generally, the time
required to obtain greater than about 80% sialylation is less than
or equal to about 48 hours.
[0011] Sialyltransferases that are useful in the methods of the
invention typically have a sialyl motif that comprises about 48-50
amino acids, within which about 40% of the amino acids are
identical to the consensus sequence RCAVVSSAG - - - DVGSKT (where -
- - indicates a variable number of amino acid residues such that
the motif is about 48-50 residues in length). Examples of
sialyltransferases that are suitable for use in the present
invention include ST3Gal III (preferably a rat ST3Gal III), ST3Gal
IV, ST3Gal I, ST6Gal I, ST3Gal V, ST6Gal II, ST6GalNAc I, ST6GalNAc
II, and ST6GalNAc III (the sialyltransferase nomemclature used
herein is as described in Tsuji et al. (1996) Glycobiology 6:
v-xiv). The methods of the invention can involve sialylation of
recombinant glycoproteins with more than one sialyltransferase; for
example, with an ST3Gal III and an ST3Gal I, or an ST3 Gal III and
an ST6 GalI, or other combinations of enzymes. The sialic acid
donor moiety used in the claimed methods is generally CMP-sialic
acid, which can be added to the reaction directly or can be
enzymatically generated in situ. The sialic acids used in a
preferred embodiment are selected from the group consisting of
NeuAc and NeuGc.
[0012] The invention also provides a glycoprotein having an altered
sialylation pattern, wherein terminal galactose residues of said
glycoprotein are sialylated using the claimed methods.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1 shows a time course of ST3Gal III-mediated
sialylation of .alpha.1-acid glycoprotein which had been treated
with neuraminidase. The percentage of terminal galactose residues
which are sialylated is plotted versus the time of reaction.
[0014] FIG. 2 shows a comparison of sialylation of
neuraminidase-treated .alpha.1-acid glycoprotein using two
different sialyltransferases, ST3Gal III and ST6 Gal I.
DETAILED DESCRIPTION
[0015] Definitions
[0016] The following abbreviations are used herein:
[0017] Ara=arabinosyl;
[0018] Fru=fructosyl;
[0019] Fuc=fucosyl;
[0020] Gal=galactosyl;
[0021] GalNAc=N-acetylgalacto;
[0022] Glc=glucosyl;
[0023] GlcNAc=N-acetylgluco;
[0024] Man=mannosyl; and
[0025] NeuAc=sialyl (typically N-acetylneuraminyl).
[0026] Oligosaccharides are considered to have a reducing end and a
non-reducing end, whether or not the saccharide at the reducing end
is in fact a reducing sugar. In accordance with accepted
nomenclature, oligosaccharides are depicted herein with the
non-reducing end on the left and the reducing end on the right. All
oligosaccharides described herein are described with the name or
abbreviation for the non-reducing saccharide (e.g., Gal), followed
by the configuration of the glycosidic bond (.alpha. or .beta.),
the ring bond, the ring position of the reducing saccharide
involved in the bond, and then the name or abbreviation of the
reducing saccharide (e.g., GlcNAc). The linkage between two sugars
may be expressed, for example, as 2,3, 2.fwdarw.3, or (2,3). Each
saccharide is a pyranose.
[0027] The term "sialic acid" refers to any member of a family of
nine-carbon carboxylated sugars. The most common member of the
sialic acid family is N-acetyl-neuraminic acid
(2-keto-5-acetamindo-3,5-dideoxy--
D-glycero-D-galactononulopyranos-1-onic acid (often abbreviated as
Neu5Ac, NeuAc, or NANA). A second member of the family is
N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which the N-acetyl
group of NeuAc is hydroxylated. A third sialic acid family member
is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al. (1986) J.
Biol. Chem. 261: 11550-11557; Kanamori et al. (1990) J. Biol. Chem.
265: 21811-21819. Also included are 9-substituted sialic acids such
as a 9-O-C.sub.1-C.sub.6 acyl-NeuSAc like 9-O-lactyl-Neu5Ac or
9-O-acetyl-NeuSAc, 9-deoxy-9-fluoro-Neu5Ac and
9-azido-9-deoxy-Neu5Ac. For review of the sialic acid family, see,
e.g., Varki (1992) Glycobiology 2: 25-40; Sialic Acids: Chemistry,
Metabolism and Function, R. Schauer, Ed. (Springer-Verlag, New York
(1992)). The synthesis and use of sialic acid compounds in a
sialylation procedure is disclosed in international application WO
92/16640, published Oct. 1, 1992.
[0028] The term "recombinant" when used with reference to a cell
indicates that the cell replicates a heterologous nucleic acid, or
expresses a peptide or protein encoded by a heterologous nucleic
acid. Recombinant cells can contain genes that are not found within
the native (non-recombinant) form of the cell. Recombinant cells
can also contain genes found in the native form of the cell wherein
the genes are modified and re-introduced into the cell by
artificial means. The term also encompasses cells that contain a
nucleic acid endogenous to the cell that has been modified without
removing the nucleic acid from the cell; such modifications include
those obtained by gene replacement, site-specific mutation, and
related techniques. A "recombinant polypeptide" is one which has
been produced by a recombinant cell.
[0029] A "heterologous sequence" or a "heterologous nucleic acid",
as used herein, is one that originates from a source foreign to the
particular host cell, or, if from the same source, is modified from
its original form. Thus, a heterologous glycoprotein gene in a
eukaryotic host cell includes a glycoprotein gene that is
endogenous to the particular host cell that has been modified.
Modification of the heterologous sequence may occur, e.g., by
treating the DNA with a restriction enzyme to generate a DNA
fragment that is capable of being operably linked to the promoter.
Techniques such as site-directed mutagenesis are also useful for
modifying a heterologous sequence.
[0030] A "subsequence" refers to a sequence of nucleic acids or
amino acids that comprise a part of a longer sequence of nucleic
acids or amino acids (e.g., polypeptide) respectively.
[0031] A "recombinant expression cassette" or simply an "expression
cassette" is a nucleic acid construct, generated recombinantly or
synthetically, with nucleic acid elements that are capable of
affecting expression of a structural gene in hosts compatible with
such sequences. Expression cassettes include at least promoters and
optionally, transcription termination signals. Typically, the
recombinant expression cassette includes a nucleic acid to be
transcribed (e.g., a nucleic acid encoding a desired polypeptide),
and a promoter. Additional factors necessary or helpful in
effecting expression may also be used as described herein. For
example, an expression cassette can also include nucleotide
sequences that encode a signal sequence that directs secretion of
an expressed protein from the host cell. Transcription termination
signals, enhancers, and other nucleic acid sequences that influence
gene expression, can also be included in an expression
cassette.
[0032] The term "isolated" is meant to refer to material which is
substantially or essentially free from components which normally
accompany the enzyme as found in its native state. Typically,
isolated molecules are at least about 80% pure, usually at least
about 90%, and preferably at least about 95% pure as measured by,
e.g., band intensity on a silver stained gel or other method for
determining purity. Protein purity or homogeneity can be indicated
by a number of means well known in the art, such as polyacrylamide
gel electrophoresis of a protein sample, followed by visualization
upon staining. For certain purposes high resolution will be needed
and HPLC or a similar means for purification utilized.
[0033] The practice of this invention can involve the construction
of recombinant nucleic acids and the expression of genes in
transfected host cells. Molecular cloning techniques to achieve
these ends are known in the art. A wide variety of cloning and in
vitro amplification methods suitable for the construction of
recombinant nucleic acids such as expression vectors are well-known
to persons of skill. Examples of these techniques and instructions
sufficient to direct persons of skill through many cloning
exercises are found in Berger and Kimmel, Guide to Molecular
Cloning Techniques, Methods in Enzymology volume 152 Academic
Press, Inc., San Diego, Calif. (Berger); and Current Protocols in
Molecular Biology, F. M. Ausubel et al., eds., Current Protocols,
ajoint venture between Greene Publishing Associates, Inc. and John
Wiley & Sons, Inc., (1994 Supplement) (Ausubel). Suitable host
cells for expression of the recombinant polypeptides are known to
those of skill in the art, and include, for example, eukaryotic
cells including insect, mammalian and fungal cells. In a preferred
embodiment, Aspergillus niger is used as the host cell.
[0034] Examples of protocols sufficient to direct persons of skill
through in vitro amplification methods, including the polymerase
chain reaction (PCR) the ligase chain reaction (LCR),
Q.beta.-replicase amplification and other RNA polymerase mediated
techniques are found in Berger, Sambrook, and Ausubel, as well as
Mullis et al. (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide
to Methods and Applications (Innis et al. eds) Academic Press Inc.
San Diego, Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1,
1990) C &EN 36-47; The Journal Of NIH Research (1991) 3: 81-94;
(Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli
et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874; Lomell et al.
(1989) J. Clin. Chem. 35: 1826; Landegren et al. (1988) Science
241: 1077-1080; Van Brunt (1990) Biotechnology 8: 291-294; Wu and
Wallace (1989) Gene 4: 560; and Barringer et al. (1990) Gene 89:
117. Improved methods of cloning in vitro amplified nucleic acids
are described in Wallace et al., U.S. Pat. No. 5,426,039.
[0035] Description of the Preferred Embodiments
[0036] The present invention provides methods for efficient in
vitro sialylation of saccharide groups attached to glycoproteins,
in particular recombinantly produced glycoproteins. For example,
the methods of the invention are useful for sialylation of
recombinantly produced therapeutic glycoproteins that are
incompletely sialylated during production in mammalian cells or
transgenic animals. The methods involve contacting the saccharide
groups with a sialyltransferase and a sialic acid donor moiety for
a sufficient time and under appropriate reaction conditions to
transfer sialic acid from the sialic acid donor moiety to the
saccharide groups. Sialyltransferases comprise a family of
glycosyltransferases that transfer sialic acid from the donor
substrate CMP-sialic acid to acceptor oligosaccharide substrates.
In preferred embodiments, the sialyltransferases used in the
methods of the invention are recombinantly produced.
[0037] The methods of the invention are useful for altering the
sialylation pattern of glycoproteins. The term "altered" refers to
the sialylation pattern of a glycoprotein as modified using the
methods of the invention being different from that observed on the
glycoprotein as produced in vivo. For example, the methods of the
invention can be used to produce a glycoprotein having a
sialylation pattern that is different from that found on the
glycoprotein when it is produced by cells of the organism to which
the glycoprotein is native. Alternatively, the methods can be used
to alter the sialylation pattern of glycoproteins that are
recombinantly produced by expression of a gene encoding the
glycoprotein in a host cell, which can be of the species from which
the glycoprotein is native, or from a different species.
[0038] Recombinant glycoproteins that have sialylation patterns
that are modified by the methods of the invention can have
important advantages over proteins that are in their native,
unaltered glycosylation state, or that are in a glycosylation state
that is less than optimal for a particular application. Such
non-optimal sialylation patterns can arise when a recombinant
glycoprotein is produced in a cell that does not have the proper
complement of glycosylation machinery to produce the desired
glycosylation pattern. The optimal or preferred glycosylation
pattern may or may not be the native glycosylation pattern of the
glycoprotein when produced in its native cell. Advantages of
optimal sialylation patterns include, for example, increased
therapeutic half-life of a glycoprotein due to reduced clearance
rate. Altering the sialylation pattern can also mask antigenic
determinants on foreign proteins, thus reducing or eliminating an
immune response against the protein. Alteration of the sialylation
of a glycoprotein-linked saccharide can also be used to target a
protein to a cell surface receptor that is specific for the altered
oligosaccharide, or to block targeting to a receptor that is
specific for the unaltered saccharide.
[0039] Proteins that can be modified by the methods of the
invention include, for example, hormones such as insulin, growth
hormones (including human growth hormone and bovine growth
hormone), tissue-type plasminogen activator (t-PA), renin, clotting
factors such as factor VIII and factor IX, bombesin, thrombin,
hemopoietic growth factor, serum albumin, receptors for hormones or
growth factors, interleukins, colony stimulating factors, T-cell
receptors, MHC polypeptides, viral antigens, glycosyltransferases,
and the like. Polypeptides of interest for recombinant expression
and subsequent modification using the methods of the invention also
include al-antitrypsin, erythropoietin, granulocyte-macrophage
colony stimulating factor, anti-thrombin III, interleukin 6,
interferon .beta., protein C, fibrinogen, among many others. This
list of polypeptides is exemplary, not exclusive. The methods are
also useful for modifying the sialylation patterns of chimeric
proteins, including, but not limited to, chimeric proteins that
include a moiety derived from an immunoglobulin, such as IgG.
[0040] The in vitro sialylation methods provided by the invention
are, unlike previously described sialylation methods, practical for
commercial-scale production of modified glycoproteins. Thus, the
claimed methods provide a practical means for large-scale
preparation of glycoproteins having altered sialylation patterns.
The methods are well suited for therapeutic glycoproteins that are
incompletely sialylated during production in mammalian cells or
transgenic animals. The processes provide an increased and
consistent level of terminal sialylation of a glycoprotein.
[0041] One way by which the methods of the invention achieve
commercial feasibility is through the use of recombinantly produced
sialyltransferases. Recombinant production enables production of
sialyltransferases in the large amounts that are required for
large-scale glycoprotein modification. Deletion of the membrane
anchoring domain of sialyltransferases, which renders
sialyltransferases soluble and thus facilitates production and
purification of large amounts of sialyltransferases, can be
accomplished by recombinant expression of a modified gene encoding
the sialyltransferase. Examples of recombinant sialyltransferases,
including those having deleted anchor domains, as well as methods
of producing recombinant sialyltransferases, are found in, for
example, U.S. Pat. No. 5,541,083. At least 15 different mammalian
sialyltransferases have been documented, and the cDNAs of thirteen
of these have been cloned to date (for the systematic nomenclature
that is used herein, see, Tsuji et al. (1996) Glycobiology 6:
v-xiv). These cDNAs can be used for recombinant production of
sialyltransferases, which can then be used in the methods of the
invention.
[0042] Commercial practicality is also provided by the methods of
the invention through the use of bacterial sialyltransferases,
either recombinantly produced or produced in the native bacterial
cells. Two bacterial sialyltransferases have been recently
reported; an ST6Gal II from Photobacterium damsela (Yamamoto et al.
(1996) J. Biochem. 120: 104-110) and an ST3Gal V from Neisseria
meningitidis (Gilbert et al. (1996) J. Biol. Chem. 271:
28271-28276). The two recently described bacterial enzymes transfer
sialic acid to the Gal.beta.1,4GlcNAc sequence on oligosaccharide
substrates. However, there are no known bacterial proteins that are
glycosylated, so it was unknown whether or not the
Gal.beta.1,4GlcNAc moiety covalently linked to a protein would
serve as an acceptor substrate for a bacterial sialyltransferase.
Table 1 shows the acceptor specificity of these and other
sialyltransferases useful in the methods of the invention.
[0043] In preferred embodiments, the methods of the invention are
commercially practical due to the use of sialyltransferases that
are capable of sialylating a high percentage of acceptor groups on
a glycoprotein using a low ratio of enzyme units to glycoprotein.
In a preferred embodiment, the desired amount of sialylation will
be obtained using about 50 mU of sialyltransferase per mg of
glycoprotein or less. More preferably, less than about 40 mU of
sialyltransferase will be used per mg of glycoprotein, even more
preferably, the ratio of sialyltransferase to glycoprotein will be
less than or equal to about 45 mU/mg, and more preferably about 25
mU/mg or less. Most preferably, the desired amount of sialylation
will be obtained using less than about 10 mU/mg sialyltransferase
per mg glycoprotein. Typical reaction conditions will have
sialyltransferase present at a range of about 5-25 mU/mg of
glycoprotein, or 10-50 mU/ml of reaction mixture with the
glycoprotein present at a concentration of at least about 2
mg/ml.
[0044] Typically, the saccharide chains on a glycoprotein having
sialylation patterns altered by the methods of the invention will
have a greater percentage of terminal galactose residues sialylated
than the unaltered glycoprotein. Preferably, greater than about 80%
of terminal galactose residues present on the glycoprotein-linked
saccharide groups will be sialylated following use of the methods.
More preferably, the methods of the invention will result in
greater than about 90% sialylation, and even more preferably
greater than about 95% sialylation of terminal galactose residues.
Most preferably, essentially 100% of the terminal galactose
residues present on the glycoprotein are sialylated following
modification using the methods of the present invention. The
methods are typically capable of achieving the desired level of
sialylation in about 48 hours or less, and more preferably in about
24 hours or less.
[0045] Preferably, for glycosylation of N-linked carbohydrates of
glycoproteins the sialyltransferase will be able to transfer sialic
acid to the sequence Gal.beta.1,4GlcNAc-, the most common
penultimate sequence underlying the terminal sialic acid on fully
sialylated carbohydrate structures. Only three of the cloned
mammalian sialyltransferases meet this acceptor specificity
requirement, and each of these have been demonstrated to transfer
sialic acid to N-linked carbohydrate groups of glycoproteins.
Examples of sialyltransferases that use Gal.beta.1,4GlcNAc as an
acceptor are shown in Table 1.
1TABLE 1 Sialyltransferases which use the Gal.beta.1,4GlcNAc
sequence as an acceptor substrate. Sialyltransferase Source
Sequence(s) formed Ref. ST6Gal I Mammalian
NeuAc.alpha.2,6Gal.beta.1,4GlcNAc- 1 ST3Gal III Mammalian
NeuAc.alpha.2,3Gal.beta.1,4GlcNAc- 1
NeuAc.alpha.2,3Gal.beta.1,3GlcNAc- ST3Gal IV Mammalian
NeuAc.alpha.2,3Gal.beta.1,4GlcNAc- 1 NeuAc.alpha.2,3Gal.beta.1,3-
GlcNAc- ST6Gal II Photobacterium NeuAc.alpha.2,6Gal.beta.1,4GlcNAc-
2 ST3Gal V N. NeuAc.alpha.2,3Gal.beta.1,4GlcNAc- 3 meningitides N.
gonorrhoeae
[0046] 1) Goochee et al. (1991) Bio/Technology 9: 1347-1355
[0047] 2) Yamamoto et al. (1996) J. Biochem. 120: 104-110
[0048] 3) Gilbert et al. (1996) J. Biol. Chem. 271: 28271-28276
[0049] The substrate specificity of the sialyltransferases is only
the first criterion an enzyme must satisfy for establishing a
method for sialylation of commercially important recombinant or
transgenic glycoproteins. The sialyltransferase must also be able
to effect sialylation efficiently and completely for a variety of
glycoproteins, and support the scale-up to the 1-10 kg of
recombinant glycoprotein with relatively low cost and
infrastructure requirements. There are no published reports that
document any of these sialyltransferases to be suitable for
establishing a practical process that meets these requirements.
[0050] An example of a sialyltransferase that is useful in the
claimed methods is ST3Gal III, which is also referred to as
.alpha.(2,3)sialyltransferase (EC 2.4.99.6). This enzyme catalyzes
the transfer of sialic acid to the Gal of a Gal.beta.1,3GlcNAc or
Gal.beta.1,4GlcNAc glycoside (see, e.g., Wen et al. (1992) J. Biol.
Chem., 267: 2101 1; Van den Eijnden et al. (1991) J. Biol. Chem.,
256: 3159) and is responsible for sialylation of asparagine-linked
oligosaccharides in glycoproteins. The sialic acid is linked to a
Gal with the formation of an .alpha.-linkage between the two
saccharides. Bonding (linkage) between the saccharides is between
the 2-position of NeuAc and the 3-position of Gal. This particular
enzyme can be isolated from rat liver (Weinstein et al. (1982) J.
Biol. Chem., 257: 13845); the human cDNA (Sasaki et al. (1993) J.
Biol. Chem. 268:22782-22787; Kitagawa & Paulson (1994) J. Biol.
Chem. 269:1394-1401) and genomic (Kitagawa et al. (1996) J. Biol.
Chem. 271:931-938) DNA sequences are known, facilitating production
of this enzyme by recombinant expression. In a preferred
embodiment, the claimed sialylation methods use a rat ST3Gal
III.
[0051] Other sialyltransferases, including those listed in Table 1,
may also be useful in an economic and efficient large scale process
for sialylation of commercially important glycoproteins. As a
simple test to find out the utility of these other enzymes, various
amounts of each enzyme (1-100 mU/mg protein) are reacted with
asialo-.alpha..sub.1 AGP (at 1-10 mg/ml) to compare the ability of
the sialyltransferase of interest to sialylate glycoproteins
relative to either bovine ST6Gal I, ST3Gal III or both
sialyltransferases. Alternatively, other glycoproteins or
glycopeptides, or N-linked oligosaccharides enzymatically released
from the peptide backbone can be used in place of
asialo-.alpha..sub.1 AGP for this evaluation. Sialyltransferases
showing an ability to sialylate N-linked oligosaccharides of
glycoproteins more efficiently than ST6Gal I may prove useful in a
practical large scale process for glycoprotein sialylation (as
illustrated for ST3Gal III in this disclosure).
[0052] The invention also provides methods of altering the
sialylation pattern of a glycoprotein by adding sialic acid in an
.alpha.2,6Gal linkage as well as the .alpha.2,3Gal linkage, both of
which are found on N-linked oligosaccharides of human plasma
glycoproteins. In this embodiment, ST3Gal III and ST6Gal I
sialyltransferases are both present in the reaction and provide
proteins having a reproducible ratio of the two linkages formed in
the resialylation reaction. Thus, a mixture of the two enzymes may
be of value if both linkages are desired in the final product.
[0053] An acceptor for the sialyltransferase will be present on the
glycoprotein to be modified by the methods of the present
invention. Suitable acceptors include, for example, galactosyl
acceptors such as Gal.beta.1,4GlcNAc, Gal.beta.1,4GalNAc,
Gal.beta.1,3 GalNAc, lacto-N-tetraose, Gal.beta.1,3GlcNAc,
Gal.beta.1,3Ara, Gal.beta.1,6GlcNAc, Gal.beta.1,4Glc (lactose), and
other acceptors known to those of skill in the art (see, e.g.,
Paulson et al. (1978) J. Biol. Chem. 253: 5617-5624). Typically,
the acceptors are included in oligosaccharide chains that are
attached to asparagine, serine, or threonine residues present in a
protein.
[0054] In one embodiment, an acceptor for the sialyltransferase is
present on the glycoprotein to be modified upon in vivo synthesis
of the glycoprotein. Such glycoproteins can be sialylated using the
claimed methods without prior modification of the glycosylation
pattern of the glycoprotein. Alternatively, the methods of the
invention can be used to alter the sialylation pattern of a
glycoprotein that has been modified prior to sialylation. For
example, to sialylate a protein that does not include a suitable
acceptor, one can modify the protein to include an acceptor by
methods known to those of skill in the art. The acceptor can be
synthesized by attaching a galactose residue to, for example, a
GlcNAc or other appropriate saccharide moiety that is linked to the
protein. Glycoprotein-linked oligosaccharides can be first
"trimmed," either in whole or in part, to expose either an acceptor
for the sialyltransferase or a moiety to which one or more
appropriate residues can be added to obtain a suitable acceptor.
Enzymes such as glycosyltransferases and endoglycosidases are
useful for the attaching and trimming reactions. The claimed
methods are also useful for synthesizing a sialic acid-terminated
saccharide moiety on a protein that is unglycosylated in its native
form. A suitable acceptor for the sialyltransferase is attached to
such proteins by methods known to those of skill in the art prior
to sialylation using the methods of the present invention. See,
e.g., U.S. Pat. No. 5,272,066 for methods of obtaining polypeptides
having suitable acceptors for sialylation.
[0055] Thus, in one embodiment, the invention provides methods for
in vitro sialylation of saccharide groups present on a glycoprotein
that first involves modifying the glycoprotein to create a suitable
acceptor. A preferred method for synthesizing an acceptor involves
use of a galactosyltransferase. The steps for these methods
include:
[0056] (a) galactosylating a compound of the formula
GlcNR'.beta.(1.fwdarw.3)Gal.beta.-OR with a galactosyltransferase
in the presence of a UDP-galactose under conditions sufficient to
form the compound:
Gal.beta.(1.fwdarw.4)GlcNR'.beta.1.fwdarw.3)Gal.beta.-OR; and
[0057] (b) sialylating the compound formed in (a) with a
sialyltransferase in the presence of a CMP derivative of a sialic
acid using an a(2,3)sialyltransferase under conditions in which
sialic acid is transferred to the non-reducing sugar to form the
compound:
NeuAc.alpha.(2.fwdarw.3)Gal.beta.(1.fwdarw.4)GlcNR'.beta.(1.fwdarw.3)Gal.-
beta.-OR. In this formula, R is an amino acid, a saccharide, an
oligosaccharide or an aglycon group having at least one carbon
atom. R' can be either acetyl or allyloxycarbonyl (Alloc). R is
linked to or is part of a glycoprotein.
[0058] The galactosylating and sialylating steps are preferably
carried out enzymatically, with the galactosylating step preferably
being carried out as part of a galactosyltransferase cycle and the
sialylating step preferably being carried out as part of a
sialyltransferase cycle. Preferred conditions and descriptions of
other species and enzymes in each of these cycles has been
described. In a preferred embodiment, the galactosylating and
sialylating steps are carried out in a single reaction mixture that
contains both sialyltransferase and galactosyltransferase. In this
embodiment, the enzymes and substrates can be combined in an
initial reaction mixture, or preferably the enzymes and reagents
for a second glycosyltransferase cycle can be added to the reaction
medium once the first glycosyltransferase cycle has neared
completion. By conducting two glycosyltransferase cycles in
sequence in a single vessel, overall yields are improved over
procedures in which an intermediate species is isolated. Moreover,
cleanup and disposal of extra solvents and by-products is
reduced.
[0059] In a preferred embodiment, the sialylation of the
glycoprotein is accomplished using a sialyltransferase cycle, which
includes a CMP-sialic acid recycling system that utilizes
CMP-sialic acid synthetase. CMP-sialic acid is relatively
expensive, so in situ synthesis of this sialic acid donor moiety
enhances the economic advantages provided by the claimed methods.
Sialyltransferase cycles are described, for example, in U.S. Pat.
No. 5,374,541. The CMP-sialic acid regenerating system used in this
embodiment comprises cytidine monophosphate (CMP), a nucleoside
triphosphate, a phosphate donor, a kinase capable of transferring
phosphate from the phosphate donor to nucleoside diphosphates and a
nucleoside monophosphate kinase capable of transferring the
terminal phosphate from a nucleoside triphosphate to CMP.
[0060] The regenerating system also employs CMP-sialic acid
synthetase, which transfers sialic acid to CTP. CMP-sialic acid
synthetase can be isolated and purified from cells and tissues
containing the synthetase enzyme by procedures well known in the
art. See, for example, Gross et al. (1987) Eur. J. Biochem., 168:
595; Vijay et al. (1975) J. Biol. Chem. 250: 164; Zapata et al.
(1989) J. Biol. Chem. 264: 14769; and Higa et al. (1985) J. Biol.
Chem. 260: 8838. The gene for this enzyme has also been sequenced.
See, Vann et al. (1987) J. Biol. Chem., 262:17556. Overexpression
of the gene has been reported for use in a gram scale synthesis of
CMP-NeuAc. See, Shames et al. (1991) Glycobiology, 1:187. This
enzyme is also commercially available.
[0061] Nucleoside triphosphates suitable for use in accordance with
the CMP-sialic acid regenerating system are adenosine triphosphate
(ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP),
guanosine triphosphate (GTP), inosine triphosphate (ITP) and
thymidine triphosphate (TTP). A preferred nucleoside triphosphate
is ATP.
[0062] Nucleoside monophosphate kinases are enzymes that catalyze
the phosphorylation of nucleoside monophosphates. Nucleoside
monophosphate kinase (NMK) or myokinase (MK; EC 2.7.4.3) used in
accordance with the CMP-sialic acid regenerating system of the
present invention are used to catalyze the phosphorylation of CMP.
NMK's are commercially available (Sigma Chem. Co., St. Louis, Mo.;
Boehringer Mannheim, Indianapolis, Ind.).
[0063] A phosphate donor and a catalytic amount of a kinase that
catalyzes the transfer of phosphate from the phosphate donor to an
activating nucleotide are also part of the CMP-sialic acid
regenerating system. The phosphate donor of the regenerating system
is a phosphorylated compound, the phosphate group of which can be
used to phosphorylate the nucleoside phosphate. The only limitation
on the selection of a phosphate donor is that neither the
phosphorylated nor the dephosphorylated forms of the phosphate
donor can substantially interfere with any of the reactions
involved in the formation of the sialylated galactosyl glycoside.
Preferred phosphate donors are phosphoenolpyruvate (PEP), creatin
phosphate, and acetyl phosphate. A particularly preferred phosphate
donor is PEP.
[0064] The selection of a particular kinase for use in a sialic
acid cycle depends upon the phosphate donor employed. When acetyl
phosphate is used as the phosphate donor, the kinase is acetyl
kinase; creatin kinase is used for a creatin phosphate donor, and
when PEP is used as the phosphate donor, the kinase is pyruvate
kinase (PK; EC 2.7.1.40). Other kinases can be employed with other
phosphate donors as is well known to those of skill in the art.
Kinases are commercially available (Sigma Chem. Co., St. Louis,
Mo.; Boehringer Mannheim, Indianapolis, Ind.).
[0065] Because of the self-contained and cyclic character of this
glycosylation method, once all the reactants and enzymes are
present, the reaction continues until the first of the
stoichiometric substrates (e.g. free Neu5Ac and PEP, or the
acceptor) is consumed.
[0066] In the sialylation cycle, CMP is converted to CDP by
nucleoside monophosphate kinase in the presence of added AT?. ATP
is catalytically regenerated from its byproduct, ADP, by pyruvate
kinase (PK) in the presence of added phosphoenolpyruvate (PEP). CDP
is further converted to CTP, which conversion is catalyzed by PK in
the presence of PEP. CTP reacts with sialic acid to form inorganic
pyrophosphate (PPi) and CMP-sialic acid, the latter reaction being
catalyzed by CMP-sialic acid synthetase. Following sialylation of
the galactosyl glycoside, the released CMP re-enters the
regenerating system to reform CDP, CTP and CMP-sialic acid. The
formed PPi is scavenged as discussed below, and forms inorganic
phosphate (Pi) as a byproduct. Pyruvate is also a byproduct.
[0067] The byproduct pyruvate can also be made use of in another
reaction in which N-acetylmannosamine (ManNAc) and pyruvate are
reacted in the presence of NeuAc aldolase (EC 4.1.3.3) to form
sialic acid. Thus, the sialic acid can be replaced by ManNAc and a
catalytic amount of NeuAc aldolase. Although NeuAc aldolase also
catalyzes the reverse reaction (NeuAc to ManNAc and pyruvate), the
produced NeuAc is irreversibly incorporated into the reaction cycle
via CMP-NeuAc catalyzed by CMP-sialic acid synthetase. This
enzymatic synthesis of sialic acid and its 9-substituted
derivatives and the use of a resulting sialic acid in a different
sialylating reaction scheme is disclosed in International
application WO 92/16640, published on Oct. 1, 1992.
[0068] As used herein, the term "pyrophosphate scavenger" refers to
substances that serve to remove inorganic pyrophosphate from a
reaction mixture of the present invention. Inorganic pyrophosphate
(PPi) is a byproduct of the preparation of CMP-Neu5Ac. Produced PPi
can feed back to inhibit other enzymes such that glycosylation is
reduced. However, PPi can be degraded enzymatically or by physical
means such as sequestration by a PPi binding substance. Preferably,
PPi is removed by hydrolysis using inorganic pyrophosphatase
(PPase; EC 3.6.1.1), a commercially available PPi catabolic enzyme
(Sigma Chem. Co., St. Louis, Mo.; Boehringer Mannheim,
Indianapolis, Ind.), and that or a similar enzyme serves as the
pyrophosphate scavenger. One method of removing PPi or Pi from the
reaction mixture is to maintain divalent metal cation concentration
in the medium. In particular, the cations and the inorganic
phosphate produced form a complex of very low solubility. By
supplementing the cations which are lost by precipitation with
pyrophosphate, the rate of reaction can be maintained and the
reactions can be taken to completion (i.e., 100% conversion).
Supplementing can be carried out continuously (e.g., by automation)
or discontinuously. When cation concentration is maintained in this
way, the transferase reaction cycle can be driven to
completion.
[0069] For glycosyltransferase cycles, the concentrations or
amounts of the various reactants used in the processes depend upon
numerous factors including reaction conditions such as temperature
and pH value, and the choice and amount of acceptor saccharides to
be glycosylated. Because the glycosylation process permits
regeneration of activating nucleotides, activated donor sugars and
scavenging of produced PPi in the presence of catalytic amounts of
the enzymes, the process is limited by the concentrations or
amounts of the stoichiometric substrates discussed before. The
upper limit for the concentrations of reactants that can be used in
accordance with the method of the present invention is determined
by the solubility of such reactants. Preferably, the concentrations
of activating nucleotides, phosphate donor, the donor sugar and
enzymes are selected such that glycosylation proceeds until the
acceptor is consumed, thus completely sialylating the saccharide
groups present on the glycoprotein.
[0070] Enzyme amounts or concentrations are expressed in activity
Units, which is a measure of the initial rate of catalysis. One
activity Unit catalyzes the formation of 1 .mu.mol of product per
minute at a given temperature (typically 37.degree. C.) and pH
value (typically 7.5). Thus, 10 Units of an enzyme is a catalytic
amount of that enzyme where 10 .mu.mols of substrate are converted
to 10 .mu.mol of product in one minute at a temperature of
37.degree. C. and a pH value of 7.5.
[0071] The above ingredients are combined by admixture in an
aqueous reaction medium (solution). That medium has a pH value of
about 6 to about 8.5. The medium is devoid of chelators that bind
enzyme cofactors such as Mg.sup.+2 or Mn.sup.+2. The selection of a
medium is based on the ability of the medium to maintain pH value
at the desired level. Thus, in some embodiments, the medium is
buffered to a pH value of about 7.5, preferably with HEPES. If a
buffer is not used, the pH of the medium should be maintained at
about 6 to 8.5, preferably about 7.2 to 7.8, by the addition of
base. A suitable base is NaOH, preferably 6 M NaOH.
[0072] The reaction medium may also comprise solubilizing
detergents (e.g., Triton or SDS) and organic solvents such as
methanol or ethanol, if necessary. The enzymes can be utilized free
in solution or can be bound to a support such as a polymer. The
reaction mixture is thus substantially homogeneous at the
beginning, although some precipitate can form during the
reaction.
[0073] The temperature at which an above process is carried out can
range from just above freezing to the temperature at which the most
sensitive enzyme denatures. That temperature range is preferably
about zero degrees C to about 45.degree. C., and more preferably at
about 20.degree. C. to about 37.degree. C.
[0074] The reaction mixture so formed is maintained for a period of
time sufficient for the desired percentage of terminal galactose
residues present on saccharide groups attached to the glycoprotein
to be sialylated. For commercial-scale preparations, the reaction
will often be allowed to proceed for about 8-240 hours, with a time
of between about 24 and 48 hours being more typical.
[0075] The following examples are offered to illustrate, but not to
limit the present invention.
EXAMPLE 1
Sialylation of Recombinant Glycoproteins Using ST3Gal III
[0076] Several glycoproteins were examined for their ability to be
sialylated by recombinant rat ST3Gal III. For each of these
glycoproteins, sialylation will be a valuable process step in the
development of the respective glycoproteins as commercial
products.
[0077] Reaction Conditions
[0078] Reaction conditions were as summarized in Table 2. The
sialyltransferase reactions were carried out for 24 hour at a
temperature between room temperature and 37.degree.. The extent of
sialylation was established by determining the amount of
.sup.14C-NeuAc incorporated into glycoprotein-linked
oligosaccharides.
[0079] Results and Discussion
[0080] The results presented in Table 2 demonstrate that a
remarkable extent of sialylation was achieved in every case,
despite low levels of enzyme used (essentially complete sialylation
was obtained based on the estimate of available terminal
galactose). Table 2 shows the amount of enzyme used per mg of
protein (mU/mg) as a basis of comparison for the various studies.
In several of the examples shown, only 7-13 mU ST3Gal III per mg of
protein was required to give essentially complete sialylation after
24 hr.
[0081] These results are in marked contrast to those reported in
detailed studies with bovine ST6Gal I where >50 mU/mg protein
gave less than 50% sialylation, and 1070 mU/mg protein gave
approximately 85-90% sialylation in 24 hr. Paulson et al. (1977) J.
Biol. Chem. 252: 2363-2371; Paulson et al. (1978) J. Biol. Chem.
253: 5617-5624. A study of rat .alpha.2,3 and .alpha.2,6
sialyltransferases by another group found that complete sialylation
of asialo-.alpha..sub.1AGP required enzyme concentrations of
150-250 mU/mg protein. Weinstein et al. (1982) J. Biol. Chem. 257:
13845-13853. These earlier studies taken together suggested that
the ST6Gal I sialyltransferase requires greater than 50 mU/mg and
up to 150 mU/mg to achieve complete sialylation.
[0082] This Example demonstrates that sialylation of recombinant
glycoproteins using the ST3 Gal III sialyltransferase requires much
less enzyme than expected. For a one kilogram scale reaction,
approximately 7,000 units of the ST3Gal III sialyltransferase would
be needed instead of 100,000-150,000 units that the earlier studies
indicated. Purification of these enzymes from natural sources is
prohibitive, with yields of only 1-10 units for a large scale
preparation after 1-2 months work. Assuming that both the ST6Gal I
and ST3Gal III sialyltransferases are produced as recombinant
sialyltransferases, with equal levels of expression of the two
enzymes being achieved, a fermentation scale 14-21 times greater
(or more) would be required for the ST6Gal I sialyltransferase
relative to the ST3Gal III sialyltransferase. For the ST6Gal I
sialyltransferase, expression levels of 0.3 U/I in yeast has been
reported. Borsig et al. (1995) Biochem. Biophys. Res. Commun. 210:
14-20. Expression levels of 1000 U/liter of the ST3Gal III
sialyltransferase have been achieved in Aspergillus niger. At
current levels of expression 300-450,000 liters of yeast
fermentation would be required to produce sufficient enzyme for
sialylation of 1 kg of glycoprotein using the ST6Gal I
sialyltransferase. In contrast, less than 10 liter fermentation of
Aspergillus niger would be required for sialylation of 1 kg of
glycoprotein using the ST3Gal III sialyltransferase. Thus, the
fermentation capacity required to produce the ST3 Gal III
sialyltransferase for a large scale sialylation reaction would be
10-100 fold less than that required for producing the ST6Gal I; the
cost of producing the sialyltransferase would be reduced
proportionately.
2TABLE 2 Sialylation of recombinant glycoproteins using the ST3Gal
III sialytransferase Reaction Conditions CMP- Analytical Results
Protein NeuAc Terminal NeuAc Total Conc. ST ST/Protein or Gal.sup.2
Incorp..sup.3 Other Protein Source (mg) (mg/ml) (mU/ml) (mU/mg)
`cycle`.sup.1 mol/mol mol/mol % Rxn.sup.4 Characteristics
ATIII.sup.5 Genzyme 8.6 4.3 210 48 cycle 1.2 1.4 117 None
Transgenics ATIII.sup.5 Genzyme 860 4.3 53 12 cycle 1.2 1.3 108
SDS-gels: protein purity Transgenics FACS: carbohydrate glycoforms
Asialo- Sigma 0.4 1.5 20 13 10 mM 8.2 9.5 116 None fetuin asialo-
PPL 0.4 0.5 20 40 20 mM 7 7.0 100 SDS-gels: protein purity
AAT.sup.6 .sup.1`Cycle` refers to generation of CMP-NeuAc `in situ`
enzymatically using standard conditions as described in the
specification (20 mM NeuAc and 2 mM CMP). The buffer was 0.1M
HEPES, pH 7.5. .sup.2Terminal (exposed) Gal content on N-linked
oligosaccharides determined by supplier, or from literature values
(fetuin, asiolo-AAAT). .sup.3NeuAc incorporated determined by
incorporation of 14C-NeuAc after separation from free radiolabeled
precursors by gel filtration. .sup.4The % Rxn refers to %
completion of the reaction based on the terminal Gal content as a
theoretical maximum. .sup.5Antithrombin III. .sup.6.alpha.1
Antitrypsin.
EXAMPLE 2
Kinetics of Sialylation of Recombinant Glycoprotein using ST3Gal
III
[0083] Reaction Conditions
[0084] Assay mixtures (total volume of 500 .mu.l) consisted of: 25
mM MES pH 6.0, 0.5% (v/v) Triton CF-54,2 mg/ml BSA, 0.04% sodium
azide, 1 mg neuraminidase treated-.alpha.1-acid glycoproteins,
sialyltransferase (2-100 mUnit/ml), and 3400 nmole of CMP-sialic
acid with a CMP-[.sup.14C]SA tracer added to follow the extent of
sialylation. The ST3Gal III was recombinantly produced, while the
ST6Gal I was purified from bovine colostrum. The concentration of
neuraminidase-treated .alpha.1-acid glycoprotein was determined by
absorption using a predetermined extinction coefficient
(.epsilon.278=0.894 for 1 mg) and by the amount of terminal
galactose as determined by the galactose dehydrogenase assay
(Wallenfels and Kurz, G. (1966) Meth. Enzymol. 9: 112-116.).
[0085] After the indicated incubation times at 37.degree. C., the
extent of sialylation of neuraminidase treated .alpha.1-acid
glycoprotein was determined by removing 50 .mu.l (10%) aliquots
from the reaction mixture and glycoprotein acceptor was
precipitated with 1 ml of 1% phosphotungstic acid in 0.5M HCl to
separate it from CMP-SA donor. The pellet was washed twice with
phosphotungstic acid followed by dissolving the pellet in 400 .mu.l
of chloroform/methanol 1:1 (v/v) of 4.degree. C. for 20 minutes.
After a final pellet was obtained by centrifugation, the
supernatant was removed and the pellet allowed to dry. The pellet
was then dissolved in 400 .mu.l of 0.2M NaCl, 0.5N NaOH at
37.degree. C. for 1 hr. The dissolved pellet was then transferred
to scintillation vials for scintillation counting. Negative
controls represented by omitting the acceptor were subtracted from
each time point.
[0086] Results and Discussion
[0087] A time course of sialylation using ST3Gal III at a
concentration of 20 mUnit/ml (10 mUnit/mg acceptor) is shown in
FIG. 1. These results demonstrate that ST3 Gal III efficiently
sialylates open galactose residues on neuraminidase-treated
.alpha.1-acid glycoprotein. In fact greater than 80% sialylation is
achieved in one hour. The achievement of greater than 80%
sialylation in one hour is significant in that recombinant
glycoproteins of therapeutic value may lose bioactivity with
extended incubation times at 37.degree. C.
[0088] It should be noted that neuraminidase treated .alpha.1-acid
glycoprotein is particularly difficult glycoprotein to completely
sialylate due to the multiple tri- and tetra-antennary N-linked
oligosaccharides. In fact, using neuraminidase treated
.alpha.1-acid glycoprotein as an acceptor ST3Gal III, is superior
to another common sialyltransferase, ST6Gal I isolated from bovine
colostrum. A comparison of the sialylation capabilities of these
two enzymes using neuraminidase treated .alpha.1-acid glycoprotein
as an acceptor is shown in FIG. 2. These results demonstrate that
ST3GalIII is superior to ST6Gal I at every time point examined,
particularly with shorter incubation times. At one hour, ST3 Gal
III had sialylated 80% of the acceptors open galactose residues,
while only 30% of the sites were saturated by ST6Gal I.
[0089] When different batches of neuraminidase treated al -acid
glycoprotein were used as acceptor utilizing similar assay
conditions, the percent saturation of open galactose ranged from
75-99% for ST3Gal III and 42-60% for ST6Gal I at 24 hours. These
results represent experiments in which ST3Gal III and ST6Gal I are
compared in parallel using identical conditions as defined above.
For these experiments, neuraminidase treated .alpha.1-acid
glycoprotein acceptor is separated from donor by gel filtration as
described previously (Weistein et al. (1982) J. Biol. Chem. 257:
13845-13853).
[0090] In each case examined, ST3Gal III sialylated the acceptor to
a level significantly greater than the extent of sialylation
achieved with ST6Gal I up to 24 hours.
[0091] In addition to examining the above mammalian
sialyltransferases, two bacterial sialyltransferases were examined
for their ability to sialylate .alpha.1-acid glycoprotein. An
unanticipated finding was that a recombinant 2,3 sialyltransferase
from Neisseria meningtidis did not transfer sialic acid to
.alpha.1-acid glycoprotein under conditions in which it sialylates
oligosaccharides containing terminal Gal.beta.1,4 such as LNnT and
lactose. In contrast, a 2,6 sialyltransferase purified from
Photobacterium damsela did efficiently incorporate sialic acid into
neuraminidase treated .alpha.-acid glycoprotein as an acceptor.
EXAMPLE III
Identification of Sialyltransferases Useful in Methods For
Practical Commercial Glycoprotein Modification
[0092] Members of the mammalian sialyltransferase gene family shown
in Table 3 below are expressed recombinantly and examined for their
ability to sialylate a variety glycoproteins in a commercially
practical manner.
3TABLE 3 Mammalian Sialyltransferases Sialyltransferase Sequences
formed ST3Gal I Neu5Ac.alpha.2,3Gal.beta.1,3GalNAc ST3Gal II
Neu5Ac.alpha.2,3Gal.beta.1,4GlcNAc ST3Gal IV
Neu5Ac.alpha.2,3Gal.beta.1,4GlcNAc Neu5Ac.alpha.2,3Gal.beta.1,3G-
lcNAc ST5GalNAc I Neu5Ac2,6GalNAc Gal.beta.1,3GalNAc(Neu5-
Ac.alpha.2,6) Gal.beta.1,3GalNAc(Neu5Ac.alpha.2,6)
Neu5Ac.alpha.2,3Gal.beta.1,3GalNAc(Neu5Ac.alpha.2,6) ST6GalNAc II
Neu5Ac2,6GalNAc Gal.beta.1,3GalNAc(Neu5Ac.alpha.2,6) ST6GalNAc III
Neu5Ac.alpha.2,3Gal.beta.1,3GalNAc(Neu5Ac.alpha.2,6)
[0093] Sialyltransferases capable of sialylating glycoproteins to a
level of at least 80% using no more than 50 mUnits/mg of acceptor
are considered "practical" for use in commercial-scale glycoprotein
modification. The analysis utilizes assay conditions that are
practical for use on a large scale, e.g., 1-10 mg/ml glycoprotein
acceptor and a sialyltransferase concentration of (2-50 mUnit/mg of
acceptor). The amount of open galactose is determined by the
galactose dehydrogenase assay (Wallenfels et al., supra.). After
appropriate incubation times at 37.degree. C., the extent of
glycoprotein is assessed by removing aliquots from the reaction
mixture and separating the glycoprotein from CMP-SA donor by
precipitation or by gel filtration.
[0094] Additionally, recombinant or purified sialyltransferases
from bacteria displayed in Table 4 below can be examined. Again the
sialyltransferase concentration do not exceed 50 mUnits/mg of
glycoprotein acceptor and glycoprotein concentrations range from
1-10 mg/ml.
4TABLE 4 Bacterial Sialyltransferases Sialyltransferase Organism
Structure formed Sialyltransferase N. meningitidis
Neu5Ac.alpha.2,3Gal.beta.1,4GlcNAc N. gonorrheae ST3Gal VI
Campylobacter jejuni Neu5Ac.alpha.2,3Gal.beta.1,4GlcNAc ST3Gal VII
Haemophilus somnus (also H. influenzae
Neu5Ac.alpha.2,3Gal.beta.1,3GlcNAc) ST3Gal VIII ST6Gal II
Photobacterium Neu5Ac.alpha.2,6Gal.beta.1,4G- lcNAc damsela
[0095] The bacterial and mammalian sialyltransferases listed in
Tables 3 and 4 are tested for their ability to fully sialylate the
recombinant or transgenically expressed glycoproteins such as those
displayed in Table 5 below. This list is not meant to be exhaustive
but instead provides examples of glycoproteins of known therapeutic
utility where complete sialylation may favorably alter the
pharmacokinetics or biological activity of the glycoprotein. The
glycoproteins used in these experiments can be produced in a
transgenic animal, or in a eukaryotic cell or cell line.
[0096] In this experiment, the extent of sialylation and type of
glycan modifying the glycoprotein of interest is examined using
standard biochemical techniques such as gel electrophoresis, HPLC
and mass spectrometry. This structural information is used to
choose sialyltransferases with the correct specificity
characteristics to completely (or as close as possible) sialylate
the glycoprotein as judged by gel electrophoresis or HPLC of the
resulting glycans. At this point the pharmacokinetics of the fully
sialylated glycoprotein can be compared with the pharmacokinetics
of the under-sialylated glycoprotein by examination in small
animals.
[0097] It is recognized that certain glycoproteins will require a
combination of sialyltransferases given the stereochemical and
regioselective nature of this class of enzymes. Therefore,
combinations of sialyltransferases are examined utilizing the
defined conditions for their potential large scale practicality in
remodeling glycoproteins. This is of particular importance when
examining glycoproteins with both N-linked as well as O-linked
glycans as well as those modified by highly branched
oligosaccharides. In this regard, sialyltransferases that display
multiple specificities such as the ST-3Gal IV and the Campylobacter
sialyltransferase may be particularly useful as stand-alone
remodeling enzymes when sialylating glycoproteins with multiple N
and O-linked glycans.
5TABLE 5 Glycoprotein sialylation candidates.
.alpha.-1-anti-trypsin Tissue plasminogen activator Erythropoietin
Granulocyte-macrophage colony stimulating factor (GMCSF)
Anti-thrombin III Human growth hormone Human interleukin 6
Interferon .beta. Protein C Fibrinogen Factor IX Factor VII Tumor
necrosis factor Tumor necrosis factor receptor protein
[0098] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference for all purposes.
Sequence CWU 1
1
* * * * *