U.S. patent application number 12/444380 was filed with the patent office on 2010-03-25 for methods for the purification of polypeptide conjugates.
This patent application is currently assigned to Novo Nordisk A/S. Invention is credited to Shawn Defrees, Kyle Kinealy.
Application Number | 20100075375 12/444380 |
Document ID | / |
Family ID | 39365174 |
Filed Date | 2010-03-25 |
United States Patent
Application |
20100075375 |
Kind Code |
A1 |
Defrees; Shawn ; et
al. |
March 25, 2010 |
METHODS FOR THE PURIFICATION OF POLYPEPTIDE CONJUGATES
Abstract
The present invention provides processes for the manufacturing
of polypeptide conjugates. In particular, the invention provides
methods for the purification of polypeptide conjugates, which
include at least one polymeric modifying groups, such as a
poly(alkylene oxide) moiety. Exemplary poly(alkylene oxide)
moieties include poly(ethylene glycol) (PEG) and poly(propylene
glycol). In an exemplary process, hydrophobic interaction
chromatography (HIC) is used to resolve different glycoforms of
glycoPEGylated polypeptides.
Inventors: |
Defrees; Shawn; (North
Wales, PA) ; Kinealy; Kyle; (Plymouth Meeting,
PA) |
Correspondence
Address: |
LEYDIG VOIT & MAYER, LTD
TWO PRUDENTIAL PLAZA, SUITE 4900, 180 NORTH STETSON AVENUE
CHICAGO
IL
60601-6731
US
|
Assignee: |
; Novo Nordisk A/S
Bagsvaerd
DK
|
Family ID: |
39365174 |
Appl. No.: |
12/444380 |
Filed: |
October 3, 2007 |
PCT Filed: |
October 3, 2007 |
PCT NO: |
PCT/US07/80353 |
371 Date: |
July 28, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60848969 |
Oct 3, 2006 |
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60864117 |
Nov 2, 2006 |
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60868057 |
Nov 30, 2006 |
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60887517 |
Jan 31, 2007 |
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60951159 |
Jul 20, 2007 |
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60955001 |
Aug 9, 2007 |
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60956468 |
Aug 17, 2007 |
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Current U.S.
Class: |
435/68.1 ;
530/402 |
Current CPC
Class: |
C12P 21/005 20130101;
Y10S 930/09 20130101; C07K 14/00 20130101; C07K 1/13 20130101; C07K
14/505 20130101; C07K 1/18 20130101; A61K 47/60 20170801; C07K 1/20
20130101 |
Class at
Publication: |
435/68.1 ;
530/402 |
International
Class: |
C12P 21/06 20060101
C12P021/06; C07K 1/10 20060101 C07K001/10 |
Claims
1. A method of making a composition comprising a first polypeptide
conjugate, said first polypeptide conjugate comprising a first
number of poly(alkylene oxide) moieties covalently linked to said
first polypeptide, said method comprising: (a) contacting a mixture
comprising said first polypeptide conjugate and a hydrophobic
interaction chromatography (HIC) medium; and (b) eluting said first
polypeptide conjugate from said hydrophobic interaction
chromatography medium, thereby making said composition comprising
said first polypeptide conjugate.
2. The method of claim 1, wherein said mixture comprises a second
polypeptide conjugate, wherein said second polypeptide conjugate
comprises a second number of poly(alkylene oxide) moieties
covalently linked to said second polypeptide, wherein said first
number and said second number are different.
3. The method of claim 2, wherein said first polypeptide and said
second polypeptide have the same amino acid sequence.
4. The method of claim 1, wherein said first polypeptide conjugate
comprises a first glycosylation pattern, said first glycosylation
pattern comprising at least one glycan residue covalently liked to
said first polypeptide, each glycan residue optionally linked to at
least one of said poly(alkylene oxide) moieties.
5. The method of claim 4, wherein each of said poly(alkylene oxide)
moieties is covalently linked to said first polypeptide via an
O-linked or N-linked glycan residue.
6. The method of claim 4, wherein said mixture comprises a third
polypeptide conjugate comprising a third number of poly(alkylene
oxide) moieties covalently linked to said third polypeptide,
wherein said third polypeptide conjugate comprises a second
glycosylation pattern, wherein said second glycosylation pattern
differs from said first glycosylation pattern of said first
polypeptide conjugate by at least one glycosyl moiety of said at
least one glycan residue.
7. (canceled)
8. The method of claim 1, wherein said HIC medium is selected from
a butyl and a phenyl resin.
9. The method of claim 1, wherein each of said poly(alkylene oxide)
moieties is a member independently selected from a poly(ethylene
glycol) moiety and a poly(propylene glycol) moiety.
10. The method of claim 9, wherein each of said poly(alkylene
oxide) moieties has a molecular weight between about 1 kDa and
about 200 kDa.
11. The method of claim 1, wherein said first polypeptide is a
therapeutic polypeptide.
12. The method of claim 1, wherein said first polypeptide is a
member selected from, von Willebrand factor (vWF) protease, Factor
VII, Factor VIII, B-domain deleted Factor VIII, vWF-Factor VIII
fusion protein having full-length Factor VIII, vWF-Factor VIII
fusion protein having B-domain deleted Factor VIII, Factor IX,
Factor X, Factor XIII.
13. (canceled)
14. (canceled)
15. (canceled)
16. (canceled)
17. The method of claim 1, wherein at least one of said
poly(alkylene oxide) moieties is covalently linked to said first
polypeptide via a glycosyl linking group, wherein said glycosyl
linking group is either covalently linked to an amino acid residue
of said first polypeptide or is covalently linked to a glycosyl
moiety of said first polypeptide.
18. The method of claim 17, wherein said glycosyl linking group is
an intact glycosyl linking group.
19. The method of claim 18, wherein said intact glycosyl linking
group is a member selected from a GlcNH moiety, a GlcNAc moiety,
and a sialic acid moiety.
20. The method of claim 1, further comprising: (c) eluting said
first polypeptide conjugate from an anion exchange chromatography
medium.
21. The method of claim 20, wherein steps (c) is performed prior to
step (a).
22. The method of claim 1, further comprising: (d) eluting said
first polypeptide conjugate from a cation exchange chromatography
medium.
23. The method of claim 22, wherein step (d) is performed after
step (b).
24. The method of claim 1, further comprising: contacting said
first polypeptide and a modified sugar nucleotide having a glycosyl
moiety covalently linked to a poly(alkylene oxide) moiety, in the
presence of a glycosyltransferase under conditions sufficient for
said glycosyltransferase to form a covalent bond between said
glycosyl moiety and said first polypeptide, thereby forming said
first polypeptide conjugate.
25. The method of claim 24, wherein said glycosyl moiety is a
sialic acid moiety and said glycosyltransferase is a
sialyltransferase.
26. (canceled)
27. The method of claim 1, said method comprising isolating said
first polypeptide conjugate from a second polypeptide conjugate
comprising a second number of poly(alkylene oxide) moieties
covalently linked to said second polypeptide, wherein said first
number is selected from 1 to 20 and said second number is selected
from 0-20, said first number and said second number being
different, said method comprising: (a) contacting a mixture
comprising said first polypeptide conjugate and said second
polypeptide conjugate with said hydrophobic interaction
chromatography (HIC) medium; and (b) eluting said first polypeptide
conjugate from said hydrophobic interaction chromatography medium,
thereby isolating said first polypeptide conjugate from said second
polypeptide conjugate.
28-85. (canceled)
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] The present application is a U.S. National Phase of PCT
Application No. PCT/US2007/080353 filed Oct. 3, 2007 and claims
priority under 35 USC 119(e) to U.S. Provisional Patent Application
No. 60/848,969, filed on Oct. 3, 2006, U.S. Provisional Patent
Application No. 60/864,117, filed on Nov. 2, 2006; U.S. Provisional
Patent Application No. 60/868,057, filed on Nov. 30, 2006; U.S.
Provisional Patent Application No. 60/887,517, filed on Jan. 31,
2007; U.S. Provisional Patent Application No. 60/951,159, filed on
Jul. 20, 2007; U.S. Provisional Patent Application No. 60/955,001,
filed on Aug. 9, 2007; and U.S. Provisional Patent Application No.
60/956,468, filed Aug. 17, 2007, each of which is incorporated
herein by reference in their entirety for all purposes.
FIELD OF THE INVENTION
[0002] The invention pertains to the field of polypeptide
manufacturing. In particular, the invention relates to processes
for the purification of polypeptide conjugates, especially those
conjugates including poly(alkylene oxide)-based modification
groups.
BACKGROUND OF THE INVENTION
[0003] The current literature contains a wealth of information
directed to polypeptide purification methodologies, which primarily
involve chromatographic approaches as well as membrane filtration
techniques. However, effective methods for the purification of
modified polypeptides (e.g., PEGylated polypeptides) are not well
known. The modification of polypeptides with polymeric moieties
causes a significant shift in the chemical and physical properties
of those polypeptides. Methods, which are useful for the
purification of non-modified polypeptides are not necessarily
effective in capturing their modified versions.
[0004] When a glycosylated or non-glycosylated polypeptide is
subjected to a chemical modification reaction, side-products may be
formed in addition to the desired modified polypeptide. In order to
isolate a desired product from a reaction mixture, the process must
not only be suitable to remove chemical reagents, but must also be
capable of removing unwanted side-products. This is especially
important when the polypeptide is to be used as a therapeutic
agent. Polypeptide modification technologies, which rely on the
specificity of enzymes, may result in a reaction product that is
characterized by improved homogeneity when compared to other
chemical methods. However, expression of a recombinant polypeptide
in a cell (e.g., bacterial, insect, yeast or mammalian cell)
typically results in a polypeptide population that, at leas to some
extend, is characterized by a variety of glycan structures.
Subsequent modification of the polypeptide, e.g., via those
glycans, results in a heterogenous product. Although remodeling
glycan structures prior to chemical or enzymatic modification of
the polypeptide can significantly improve the quality of the
product, a certain degree of heterogeneity remains. Hence, a need
exists for production processes designed to isolate a desired
polypeptide conjugate from a reaction mixture that may not only
contain chemical reagents (e.g., those derived from unreacted
modifying groups) and/or catalytic enzymes, but may also include
polypeptide conjugate by-products. The present invention addresses
these and other needs.
SUMMARY OF THE INVENTION
[0005] The present invention provides processes for the isolation
(e.g., large-scale purification) of polypeptide conjugates. The
polypeptide conjugates of the present invention include a
polypeptide that is modified with a modifying group, such as a
polymer. Exemplary polymers include water-soluble polymers. The
methods of the invention are particularly useful for the isolation
of polypeptide conjugates that include poly(alkylene oxide)-based
polymers, such as poly(ethylene glycol) and poly(propylene glycol).
While reverse-phase (RP) chromatography can be used to purify
polypeptides that are derivatized with such highly polar,
water-soluble polymers, the technique is not desirable because it
requires the use of water-soluble organic solvents, such as
acetonitrile. Organic solvents, especially in large-scale processes
are not only associated with environmental concerns, but can also
effect the chemical stability of the purified polypeptide
conjugate. Therefore, process steps that rely on aqueous solutions
are generally preferred. Hence, in one embodiment, the current
invention provides methods that allow for the isolation of
polypeptide conjugates essentially without the use of organic
solvents.
[0006] An exemplary method of the invention involves at least one
chromatographic procedure that is effective in separating
polypeptide conjugates having at least one poly(alkylene oxide)
moiety from other components of a mixture. The methods of the
invention can be used to isolate such polypeptide conjugate from
any mixture. In one example, the mixture is a reaction mixture
(e.g., the product of a chemical PEGylation reaction or an
ezymatically catalyzed PEGylation reaction, e.g., glycoPEGylation
reaction) and may optionally include other polypeptide conjugates.
Preferred methods of the invention utilize hydrophobic interaction
chromatography (HIC) media. In one embodiment, HIC is used in
conjunction with at least one additional chromatography step
selected from anion exchange chromatography, mixed-mode
chromatography, cation exchange chromatography and hydroxyapatite
or fluoroapatite chromatography. In another embodiment, HIC is used
in conjunction with at least one of anion exchange chromatography,
mixed-mode chromatography and cation exchange chromatography. The
inventors have discovered that HIC in conjunction with cation
chromatography represents an efficient method for the resolution of
polypeptide conjugates that include at least one poly(alkylene
oxide moiety). In particular, it was discovered that HIC, followed
by cation exchange can resolve EPO-PEG.sub.3 species from
EPO-PEG.sub.2 species. In one example, HIC in conjunction with
cation exchange provided a composition of purified EPO-[PEG(10
kDa)].sub.3 having a very low residual concentration of EPO-[PEG(10
kDa)].sub.2.
[0007] An exemplary method of the invention that includes anion
exchange and cation exchange chromatography in addition to HIC is
outlined in FIG. 1. In one embodiment, the methods of the invention
are useful for the separation of different glycoforms of a
polypeptide conjugate, especially those glycoforms distinguished by
the number of poly(alkylene oxide) moieties that are linked to the
polypeptide. Unwanted glycoforms may be formed as by-products under
the reaction conditions used to form the desired polypeptide
conjugate.
[0008] Hence, in a first aspect, the invention provides a method of
making a composition that includes a first polypeptide conjugate,
the first polypeptide conjugate having a first number of
poly(alkylene oxide) moieties covalently linked to the first
polypeptide. The method includes: (a) contacting a mixture
containing the first polypeptide conjugate with a hydrophobic
interaction chromatography (HIC) medium; and (b) eluting the first
polypeptide conjugate from the HIC medium. In one example according
to this aspect, the mixture includes a second polypeptide
conjugate, wherein the second polypeptide conjugate has a second
number of poly(alkylene oxide) moieties covalently linked to the
second polypeptide, wherein the first number and the second number
are different. For example, the first polypeptide conjugate
includes 3 poly(alkylene oxide) moieties, while the second
polypeptide conjugate includes either 0, 1, 2 or 4 poly(alkylene
oxide) moieties. In one example, the poly(alkylene oxide) is
poly(ethylene glycol) (PEG).
[0009] In a second aspect, the invention provides a method of
isolating a first polypeptide conjugate including a first number of
poly(alkylene oxide) moieties covalently linked to a first
polypeptide, from a second polypeptide conjugate that includes a
second number of poly(alkylene oxide) moieties covalently linked to
a second polypeptide, wherein the first number is selected from 1
to 20 and the second number is selected from 0-20, the first number
and the second number being different. The method includes: (a)
contacting a mixture containing the first polypeptide conjugate and
the second polypeptide conjugate with a hydrophobic interaction
chromatography (HIC) medium; and (b) eluting the first polypeptide
conjugate from said hydrophobic interaction chromatography medium.
In one example according to this aspect, the first polypeptide
conjugate includes 3 poly(alkylene oxide) moieties, while the
second polypeptide conjugate includes 0, 1, 2, 4, 5, 6 or 7
poly(alkylene oxide) moieties.
[0010] In one example according to any of the above embodiments,
the first polypeptide and the second polypeptide have the same
amino acid sequence. In another example according to any of the
above embodiments, both the first and the second polypeptide are
EPO.
[0011] In a third aspect, the invention provides a method of
forming a composition that contains a first erythropoietin (EPO)
conjugate, wherein the first EPO conjugate includes a first number
of poly(alkylene oxide) moieties covalently linked to an EPO
polypeptide. The method includes: (a) contacting a mixture
containing the first EPO conjugate with an anion exchange medium;
(b) eluting the first EPO conjugate from the anion exchange medium,
forming a first eluate including the first EPO conjugate; (c)
contacting the first eluate with a hydrophobic interaction
chromatography (HIC) medium; and (d) eluting the first EPO
conjugate from the hydrophobic interaction chromatography medium.
The method may further include (e.g., after step d): (e) eluting
the first EPO conjugate from a cation exchange chromatography
medium.
[0012] In one embodiment, the method further includes forming the
polypeptide conjugate either chemically or through enzymatically
catalyzed glycomodification (e.g., glycoPEGylation using a
glycosyltransferase and an appropriate glycosyl donor molecule,
such as a modified sugar nucleotide). GlycoPEGylation methods are
art-recognized; see for example, WO 03/031464 to DeFrees et al. or
WO 04/99231, the disclosures of which are incorporated herein by
reference in their entirety.
[0013] The invention further provides compositions, which are made
by the methods of the invention as well as pharmaceutical
formulations including the composition of the invention. In
addition, the invention provides methods of treatment utilizing the
compositions of the invention.
[0014] Other objects and advantages of the invention will be
apparent to those of skill in the art from the detailed description
that follows.
DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is an overall view of an exemplary polypeptide
conjugate purification process according to a method of the
invention. The diafiltration/ultrafiltration step following
hydrophobic interaction chromatography (HIC) is optional.
[0016] FIG. 2A is an exemplary chromatogram showing the resolution
of tri-PEGylated EPO from mono-, di-, tri- and tetra-PEGylated EPO
glycoforms using hydrophobic interaction chromatography with Phenyl
650S as the separation medium.
[0017] FIG. 2B is an expanded view of the EPO-(SA-PEG-10
kDa).sub.2, EPO-(SA-PEG-10 kDa).sub.3 and EPO-(SA-PEG-10 kDa).sub.4
elution peaks of the chromatogram in FIG. 2A. The letters (E), (F)
and (G) indicate fractions that were pooled, wherein (F) indicates
the elution of EPO-(SA-PEG-10 kDa).sub.3.
[0018] FIG. 3A is a scheme of an exemplary EPO polypeptide
conjugate of the invention having an insect-specific glycosylation
pattern that includes three N-linked, monoantennary glycan residues
covalently linked to amino acid residues N24, N38 and N83. Each
glycan residue is covalently linked to a 10 kDa PEG moiety via a
terminal galactose (Gal) moiety. FIG. 3A also includes an exemplary
reaction scheme, which can be used to synthesize the EPO conjugate.
The substrate for the enzymatically catalyzed conversions is an EPO
polypeptide, which includes at least one glycan residue having a
trimannosyl moiety. In a first step, an N-acetyl glucosamine
transferase (GnT-1) is used, which adds a GlcNAc moiety to only one
of the terminal mannose moieties. In the second step, a Gal moiety
is linked to the newly added GlcNAc moiety using a galactosyl
transferase (GalT-1) forming a terminal -GlcNAc-Gal moiety. The
first and the second step maybe performed in the same reaction
vessel. In the third step, a sialic acid moiety that is modified
with a PEG moiety is linked to the terminal Gal moiety using a
sialyl transferase (ST3Gal3).
[0019] FIG. 3B is a representation of an exemplary composition of
the invention that includes various glycoforms of an exemplary
polypeptide conjugate (e.g., EPO conjugate). Each glycoform is
distinguished from other glycoforms by the number of PEG moieties
that are covalently linked to the polypeptide, or by the structure
of the glycans through which the PEG moieties are linked to the
polypeptide. Shown percentage values are exemplary.
[0020] FIG. 4A is a reverse phase (RP) HPLC chromatogram of an
exemplary glycoPEGylation reaction mixture containing
EPO-(SA-PEG-10 kDa).sub.1-4 performed at a 25 mg scale. A Zorbax
300SB-C3 (150.times.2.1 mm, 5 micron) column was used in the
analysis. The following eluants were used: 0.1% TFA in water
(Buffer A) and 0.09% TFA in CAN (Buffer B). The gradient was 42-55%
B in 14 min followed by 55-95% B in 2 min. The flow rate was 0.6
mL/min. Absorption was measured at 214 nm. The numbered peaks
represent: (1) Mono-PEG-EPO=EPO-(SA-PEG-10 kDa).sub.1; (2)
di-PEG-EPO=EPO-(SA-PEG-10 kDa).sub.2; (3)
tri-PEG-EPO=EPO-(SA-PEG-10 kDa).sub.3 and (4)
tetra-PEG-EPO=EPO-(SA-PEG-10 kDa).sub.4.
[0021] FIG. 4B is a reverse phase (RP) HPLC chromatogram of an
exemplary composition of the invention containing purified
EPO-(SA-PEG-10 kDa).sub.3 as the major component, the composition
obtained using a method of the invention. The numbered peaks
represent: (1) tri-PEG-EPO=EPO-(SA-PEG-10 kDa).sub.3 and (2)
di-PEG-EPO=EPO-(SA-PEG-10 kDa).sub.2.
[0022] FIG. 5 is a schematic representation of exemplary
glycopegylated EPO isoforms isolated from Chinese Hamster Ovary
cells. A. An exemplary 40 kilodaton O-linked pegylated glycoform.
B: One of several 30 kilodatton N-linked pegylated glycoforms. The
modified sialic acid moiety comprising the PEG molecule may occur
on any one or more of any of the branches of the N-linked glycosyl
residue. Furthermore the illustration is exemplary in that any
glycosylated EPO molecule may comprise any mixture of mono-, bi-
tri-, or tetra-antennary N-linked glycosyl residues and any one or
more of the branches may further comprise a modified sialic acid
moiety.
DETAILED DESCRIPTION OF THE INVENTION
Abbreviations
[0023] PEG, poly(ethyleneglycol); PPG, poly(propyleneglycol); Ara,
arabinosyl; Fru, fructosyl; Fuc, fucosyl; Gal, galactosyl; GalNAc,
N-acetylgalactosaminyl; Glc, glucosyl; GlcNAc,
N-acetylglucosaminyl; Man, mannosyl; ManAc, mannosaminyl acetate;
Xyl, xylosyl; and NeuAc, sialyl (N-acetylneuraminyl); M6P,
mannose-6-phosphate; BEVS, baculovirus expression vector system;
CV, column volume; NTU, nominal turbidity units; vvm,
volume/volume/min.
DEFINITIONS
[0024] Unless defined otherwise, all technical and scientific terms
used herein generally have the same meaning as commonly understood
by one of ordinary skill in the art to which this invention
belongs. Generally, the nomenclature used herein and the laboratory
procedures in cell culture, molecular genetics, organic chemistry
and nucleic acid chemistry and hybridization are those well known
and commonly employed in the art. Standard techniques are used for
nucleic acid and peptide synthesis. The techniques and procedures
are generally performed according to conventional methods in the
art and various general references (see generally, Sambrook et al.
MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. (1989) Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is
incorporated herein by reference), which are provided throughout
this document. The nomenclature used herein and the laboratory
procedures in analytical chemistry, and organic synthetic described
below are those well known and commonly employed in the art.
Standard techniques, or modifications thereof, are used for
chemical syntheses and chemical analyses.
[0025] All oligosaccharides described herein are described with the
name or abbreviation for the non-reducing saccharide (i.e., Gal),
followed by the configuration of the glycosidic bond (.alpha. or
.beta.), the ring bond (1 or 2), the ring position of the reducing
saccharide involved in the bond (2, 3, 4, 6 or 8), and then the
name or abbreviation of the reducing saccharide (i.e., GlcNAc).
Each saccharide is preferably a pyranose. For a review of standard
glycobiology nomenclature see, Essentials of Glycobiology Varki et
al. eds. CSHL Press (1999).
[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.
[0027] Where substituent groups are specified by their conventional
chemical formulae, written from left to right, they equally
encompass the chemically identical substituents, which would result
from writing the structure from right to left, e.g., --CH.sub.2O--
is intended to also recite --OCH.sub.2--.
[0028] The term "alkyl" by itself or as part of another
substituent, means, unless otherwise stated, a straight or branched
chain, or cyclic (i.e., cycloalkyl)hydrocarbon radical, or
combination thereof, which may be fully saturated, mono- or
polyunsaturated and can include di-(e.g., alkylene) and multivalent
radicals, having the number of carbon atoms designated (i.e.
C.sub.1-C.sub.10 means one to ten carbons). Examples of saturated
hydrocarbon radicals include, but are not limited to, groups such
as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,
sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl,
homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl,
n-octyl, and the like. An unsaturated alkyl group is one having one
or more double bonds or triple bonds. Examples of unsaturated alkyl
groups include, but are not limited to, vinyl, 2-propenyl, crotyl,
2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl,
3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the
higher homologs and isomers. The term "alkyl," unless otherwise
noted, is also meant to include those derivatives of alkyl defined
in more detail below, such as "heteroalkyl." Alkyl groups that are
limited to hydrocarbon groups are termed "homoalkyl".
[0029] The term "alkylene" by itself or as part of another
substituent means a divalent radical derived from an alkane, as
exemplified, but not limited, by
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--, and further includes those
groups described below as "heteroalkylene." Typically, an alkyl (or
alkylene) group will have from 1 to 24 carbon atoms, with those
groups having 10 or fewer carbon atoms being preferred in the
present invention. A "lower alkyl" or "lower alkylene" is a shorter
chain alkyl or alkylene group, generally having eight or fewer
carbon atoms.
[0030] The terms "alkoxy," "alkylamino" and "alkylthio" (or
thioalkoxy) are used in their conventional sense, and refer to
those alkyl groups attached to the remainder of the molecule via an
oxygen atom, an amino group, or a sulfur atom, respectively.
[0031] The term "heteroalkyl," by itself or in combination with
another term, means, unless otherwise stated, a stable straight or
branched chain, or cyclic hydrocarbon radical, or combinations
thereof, consisting of the stated number of carbon atoms and at
least one heteroatom selected from the group consisting of O, N, Si
and S, and wherein the nitrogen and sulfur atoms may optionally be
oxidized and the nitrogen heteroatom may optionally be quaternized.
The heteroatom(s) O, N and S and Si may be placed at any interior
position of the heteroalkyl group or at the position at which the
alkyl group is attached to the remainder of the molecule. Examples
include, but are not limited to, --CH.sub.2--CH.sub.2--O--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--CH.sub.3,
--CH.sub.2--CH.sub.2--N(CH.sub.3)--CH.sub.3,
--CH.sub.2--S--CH.sub.2--CH.sub.3, --CH.sub.2--CH.sub.2,
--S(O)--CH.sub.3, --CH.sub.2--CH.sub.2--S(O).sub.2--CH.sub.3,
--CH.dbd.CH--O--CH.sub.3, --Si(CH.sub.3).sub.3,
--CH.sub.2--CH.dbd.N--OCH.sub.3, and
CH.dbd.CH--N(CH.sub.3)--CH.sub.3. Up to two heteroatoms may be
consecutive, such as, for example, --CH.sub.2--NH--OCH.sub.3 and
CH.sub.2--O--Si(CH.sub.3).sub.3. Similarly, the term
"heteroalkylene" by itself or as part of another substituent means
a divalent radical derived from heteroalkyl, as exemplified, but
not limited by, --CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2-- and
CH.sub.2--S--CH.sub.2--CH.sub.2--NH--CH.sub.2--. For heteroalkylene
groups, heteroatoms can also occupy either or both of the chain
termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,
alkylenediamino, and the like). Still further, for alkylene and
heteroalkylene linking groups, no orientation of the linking group
is implied by the direction in which the formula of the linking
group is written. For example, the formula --CO.sub.2R'--
represents both --C(O)OR' and --OC(O)R'.
[0032] The terms "cycloalkyl" and "heterocycloalkyl", by themselves
or in combination with other terms, represent, unless otherwise
stated, cyclic versions of "alkyl" and "heteroalkyl", respectively.
Additionally, for heterocycloalkyl, a heteroatom can occupy the
position at which the heterocycle is attached to the remainder of
the molecule. Examples of cycloalkyl include, but are not limited
to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl,
cycloheptyl, and the like. Examples of heterocycloalkyl include,
but are not limited to, 1-(1,2,5,6-tetrahydropyridyl),
1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,
3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,
tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,
2-piperazinyl, and the like.
[0033] The terms "halo" or "halogen," by themselves or as part of
another substituent, mean, unless otherwise stated, a fluorine,
chlorine, bromine, or iodine atom. Additionally, terms such as
"haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl.
For example, the term "halo(C.sub.1-C.sub.4)alkyl" is mean to
include, but not be limited to, trifluoromethyl,
2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the
like.
[0034] The term "aryl" means, unless otherwise stated, a
polyunsaturated, aromatic, substituent that can be a single ring or
multiple rings (preferably from 1 to 3 rings), which are fused
together or linked covalently. The term "heteroaryl" refers to aryl
groups (or rings) that contain from one to four heteroatoms
selected from N, O, S, Si and B, wherein the nitrogen and sulfur
atoms are optionally oxidized, and the nitrogen atom(s) are
optionally quaternized. A heteroaryl group can be attached to the
remainder of the molecule through a heteroatom. Non-limiting
examples of aryl and heteroaryl groups include phenyl, 1-naphthyl,
2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,
3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,
4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl,
4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,
2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl,
4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl,
2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,
2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.
Substituents for each of the above noted aryl and heteroaryl ring
systems are selected from the group of acceptable substituents
described below.
[0035] For brevity, the term "aryl" when used in combination with
other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both
aryl and heteroaryl rings as defined above. Thus, the term
"arylalkyl" is meant to include those radicals in which an aryl
group is attached to an alkyl group (e.g., benzyl, phenethyl,
pyridylmethyl and the like) including those alkyl groups in which a
carbon atom (e.g., a methylene group) has been replaced by, for
example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl,
3-(1-naphthyloxy)propyl, and the like).
[0036] Each of the above terms (e.g., "alkyl," "heteroalkyl,"
"aryl" and "heteroaryl") are meant to include both substituted and
unsubstituted forms of the indicated radical. Preferred
substituents for each type of radical are provided below.
[0037] Substituents for the alkyl and heteroalkyl radicals
(including those groups often referred to as alkylene, alkenyl,
heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are
generically referred to as "alkyl group substituents," and they can
be one or more of a variety of groups selected from, but not
limited to: substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heterocycloalkyl, --OR', .dbd.O, .dbd.NR', .dbd.N--OR', --NR'R'',
--SR', -halogen, --SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R',
--CONR'R'', --OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R''',
--NR''C(O).sub.2R', --NR--C(NR'R''R''').dbd.NR'''',
--NR--C(NR'R'').dbd.NR''', --S(O)R', --S(O).sub.2R',
--S(O).sub.2NR'R'', --NRSO.sub.2R', --CN and NO.sub.2 in a number
ranging from zero to (2 m'+1), where m' is the total number of
carbon atoms in such radical. R', R'', R''' and R'''' each
preferably independently refer to hydrogen, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g.,
aryl substituted with 1-3 halogens, substituted or unsubstituted
alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a
compound of the invention includes more than one R group, for
example, each of the R groups is independently selected as are each
R', R'', R''' and R'''' groups when more than one of these groups
is present. When R' and R'' are attached to the same nitrogen atom,
they can be combined with the nitrogen atom to form a 5-, 6-, or
7-membered ring. For example, --NR'R'' is meant to include, but not
be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above
discussion of substituents, one of skill in the art will understand
that the term "alkyl" is meant to include groups including carbon
atoms bound to groups other than hydrogen groups, such as haloalkyl
(e.g., --CF.sub.3 and --CH.sub.2CF.sub.3) and acyl (e.g.,
--C(O)CH.sub.3, --C(O)CF.sub.3, --C(O)CH.sub.2OCH.sub.3, and the
like).
[0038] Similar to the substituents described for the alkyl radical,
substituents for the aryl and heteroaryl groups are generically
referred to as "aryl group substituents." The substituents are
selected from, for example: substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted heterocycloalkyl, --OR', .dbd.O,
.dbd.NR', .dbd.N--OR', --NR'R'', --SR', -halogen, --SiR'R''R''',
--OC(O)R', --C(O)R', --CO.sub.2R', --CONR'R'', --OC(O)NR'R'',
--NR''C(O)R', --NR'--C(O)NR''R''', --NR''C(O).sub.2R',
--NR--C(NR'R''R''').dbd.NR'''', --NR--C(NR'R'').dbd.NR''',
--S(O)R', --S(O).sub.2R', --S(O).sub.2NR'R'', --NRSO.sub.2R', --CN
and NO.sub.2, --R', --N.sub.3, --CH(Ph).sub.2,
fluoro(C.sub.1-C.sub.4)alkoxy, and fluoro(C.sub.1-C.sub.4)alkyl, in
a number ranging from zero to the total number of open valences on
the aromatic ring system; and where R', R'', R''' and R'''' are
preferably independently selected from hydrogen, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted aryl and substituted or unsubstituted
heteroaryl. When a compound of the invention includes more than one
R group, for example, each of the R groups is independently
selected as are each R', R'', R''' and R''''' groups when more than
one of these groups is present.
[0039] Two of the substituents on adjacent atoms of the aryl or
heteroaryl ring may optionally be replaced with a substituent of
the formula -T-C(O)--(CRR').sub.q--U--, wherein T and U are
independently --NR--, --O--, --CRR'-- or a single bond, and q is an
integer of from 0 to 3. Alternatively, two of the substituents on
adjacent atoms of the aryl or heteroaryl ring may optionally be
replaced with a substituent of the formula
-A-(CH.sub.2).sub.r--B--, wherein A and B are independently
--CRR'--, --O--, --NR--, --S--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2NR'-- or a single bond, and r is an integer of from 1
to 4. One of the single bonds of the new ring so formed may
optionally be replaced with a double bond. Alternatively, two of
the substituents on adjacent atoms of the aryl or heteroaryl ring
may optionally be replaced with a substituent of the formula
--(CRR').sub.s--X--(CR''R''').sub.d--, where s and d are
independently integers of from 0 to 3, and X is --O--, --NR'--,
--S--, --S(O)--, --S(O).sub.2--, or S(O).sub.2NR'--. The
substituents R, R', R'' and R''' are preferably independently
selected from hydrogen or substituted or unsubstituted
(C.sub.1-C.sub.6)alkyl.
[0040] As used herein, the term "acyl" describes a substituent
containing a carbonyl residue, C(O)R. Exemplary species for R
include H, halogen, alkoxy, substituted or unsubstituted alkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, and substituted or unsubstituted heterocycloalkyl.
[0041] As used herein, the term "fused ring system" means at least
two rings, wherein each ring has at least 2 atoms in common with
another ring. "Fused ring systems may include aromatic as well as
non aromatic rings. Examples of "fused ring systems" are
naphthalenes, indoles, quinolines, chromenes and the like.
[0042] As used herein, the term "heteroatom" includes oxygen (O),
nitrogen (N), sulfur (S), silicon (Si) and boron (B).
[0043] The symbol "R" is a general abbreviation that represents a
substituent group. Exemplary substituent groups include substituted
or unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, and substituted or unsubstituted heterocycloalkyl
groups.
[0044] The term "pharmaceutically acceptable salts" includes salts
of the active compounds which are prepared with relatively nontoxic
acids or bases, depending on the particular substituents found on
the compounds described herein. When compounds of the present
invention contain relatively acidic functionalities, base addition
salts can be obtained by contacting the neutral form of such
compounds with a sufficient amount of the desired base, either neat
or in a suitable inert solvent. Examples of pharmaceutically
acceptable base addition salts include sodium, potassium, calcium,
ammonium, organic amino, or magnesium salt, or a similar salt. When
compounds of the present invention contain relatively basic
functionalities, acid addition salts can be obtained by contacting
the neutral form of such compounds with a sufficient amount of the
desired acid, either neat or in a suitable inert solvent. Examples
of pharmaceutically acceptable acid addition salts include those
derived from inorganic acids like hydrochloric, hydrobromic,
nitric, carbonic, monohydrogencarbonic, phosphoric,
monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,
monohydrogensulfuric, hydriodic, or phosphorous acids and the like,
as well as the salts derived from relatively nontoxic organic acids
like acetic, propionic, isobutyric, maleic, malonic, benzoic,
succinic, suberic, fumaric, lactic, mandelic, phthalic,
benzenesulfonic, p-tolylsulfonic, citric, tartaric,
methanesulfonic, and the like. Also included are salts of amino
acids such as arginate and the like, and salts of organic acids
like glucuronic or galactunoric acids and the like (see, for
example, Berge et al., Journal of Pharmaceutical Science, 66: 1-19
(1977)). Certain specific compounds of the present invention
contain both basic and acidic functionalities that allow the
compounds to be converted into either base or acid addition
salts.
[0045] The neutral forms of the compounds are preferably
regenerated by contacting the salt with a base or acid and
isolating the parent compound in the conventional manner. The
parent form of the compound differs from the various salt forms in
certain physical properties, such as solubility in polar solvents,
but otherwise the salts are equivalent to the parent form of the
compound for the purposes of the present invention.
[0046] In addition to salt forms, the present invention provides
compounds, which are in a prodrug form. Prodrugs of the compounds
described herein are those compounds that readily undergo chemical
changes under physiological conditions to provide the compounds of
the present invention. Additionally, prodrugs can be converted to
the compounds of the present invention by chemical or biochemical
methods in an ex vivo environment. For example, prodrugs can be
slowly converted to the compounds of the present invention when
placed in a transdermal patch reservoir with a suitable enzyme or
chemical reagent.
[0047] Certain compounds of the present invention can exist in
unsolvated forms as well as solvated forms, including hydrated
forms. In general, the solvated forms are equivalent to unsolvated
forms and are encompassed within the scope of the present
invention. Certain compounds of the present invention may exist in
multiple crystalline or amorphous forms. In general, all physical
forms are equivalent for the uses contemplated by the present
invention and are intended to be within the scope of the present
invention.
[0048] Certain compounds of the present invention possess
asymmetric carbon atoms (optical centers) or double bonds; the
racemates, diastereomers, geometric isomers and individual isomers
are encompassed within the scope of the present invention.
[0049] The compounds of the invention may be prepared as a single
isomer (e.g., enantiomer, cis-trans, positional, diastereomer) or
as a mixture of isomers. In a preferred embodiment, the compounds
are prepared as substantially a single isomer. Methods of preparing
substantially isomerically pure compounds are known in the art. For
example, enantiomerically enriched mixtures and pure enantiomeric
compounds can be prepared by using synthetic intermediates that are
enantiomerically pure in combination with reactions that either
leave the stereochemistry at a chiral center unchanged or result in
its complete inversion. Alternatively, the final product or
intermediates along the synthetic route can be resolved into a
single stereoisomer. Techniques for inverting or leaving unchanged
a particular stereocenter, and those for resolving mixtures of
stereoisomers are well known in the art and it is well within the
ability of one of skill in the art to choose and appropriate method
for a particular situation. See, generally, Furniss et al. (eds.),
VOGEL'S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY 5.sup.TH ED.,
Longman Scientific and Technical Ltd., Essex, 1991, pp. 809-816;
and Heller, Acc. Chem. Res. 23: 128 (1990).
[0050] The graphic representations of racemic, ambiscalemic and
scalemic or enantiomerically pure compounds used herein are taken
from Maehr, J. Chem. Ed., 62: 114-120 (1985): solid and broken
wedges are used to denote the absolute configuration of a chiral
element; wavy lines indicate disavowal of any stereochemical
implication which the bond it represents could generate; solid and
broken bold lines are geometric descriptors indicating the relative
configuration shown but not implying any absolute stereochemistry;
and wedge outlines and dotted or broken lines denote
enantiomerically pure compounds of indeterminate absolute
configuration.
[0051] The terms "enantiomeric excess" and diastereomeric excess"
are used interchangeably herein. Compounds with a single
stereocenter are referred to as being present in "enantiomeric
excess," those with at least two stereocenters are referred to as
being present in "diastereomeric excess."
[0052] The compounds of the present invention may also contain
unnatural proportions of atomic isotopes at one or more of the
atoms that constitute such compounds. For example, the compounds
may be radiolabeled with radioactive isotopes, such as for example
tritium (.sup.3H), iodine-125 (.sup.125I) or carbon-14 (.sup.14C).
All isotopic variations of the compounds of the present invention,
whether radioactive or not, are intended to be encompassed within
the scope of the present invention.
[0053] "Reactive functional group," as used herein refers to groups
including, but not limited to, olefins, acetylenes, alcohols,
phenols, ethers, oxides, halides, aldehydes, ketones, carboxylic
acids, esters, amides, cyanates, isocyanates, thiocyanates,
isothiocyanates, amines, hydrazines, hydrazones, hydrazides, diazo,
diazonium, nitro, nitriles, mercaptans, sulfides, disulfides,
sulfoxides, sulfones, sulfonic acids, sulfinic acids, acetals,
ketals, anhydrides, sulfates, sulfenic acids isonitriles, amidines,
imides, imidates, nitrones, hydroxylamines, oximes, hydroxamic
acids thiohydroxamic acids, allenes, ortho esters, sulfites,
enamines, ynamines, ureas, pseudoureas, semicarbazides,
carbodiimides, carbamates, imines, azides, azo compounds, azoxy
compounds, and nitroso compounds. Reactive functional groups also
include those used to prepare bioconjugates, e.g.,
N-hydroxysuccinimide esters, maleimides and the like. Methods to
prepare each of these functional groups are well known in the art
and their application or modification for a particular purpose is
within the ability of one of skill in the art (see, for example,
Sandler and Karo, eds. ORGANIC FUNCTIONAL GROUP PREPARATIONS,
Academic Press, San Diego, 1989).
[0054] As used herein, "pharmaceutically acceptable carrier"
includes any material, which when combined with the conjugate
retains the conjugates' activity and is non-reactive with the
subject's immune systems. "Pharmaceutically acceptable carrier"
includes solids and liquids, such as vehicles, diluents and
solvents. Examples include, but are not limited to, any of the
standard pharmaceutical carriers such as a phosphate buffered
saline solution, water, emulsions such as oil/water emulsion, and
various types of wetting agents. Other carriers may also include
sterile solutions, tablets including coated tablets and capsules.
Typically such carriers contain excipients such as starch, milk,
sugar, certain types of clay, gelatin, stearic acid or salts
thereof, magnesium or calcium stearate, talc, vegetable fats or
oils, gums, glycols, or other known excipients. Such carriers may
also include flavor and color additives or other ingredients.
Compositions comprising such carriers are formulated by well known
conventional methods.
[0055] As used herein, "administering" means oral administration,
administration as a suppository, topical contact, intravenous,
intraperitoneal, intramuscular, intralesional, or subcutaneous
administration, administration by inhalation, or the implantation
of a slow-release device, e.g., a mini-osmotic pump, to the
subject. Adminsitration is by any route including parenteral and
transmucosal (e.g., oral, nasal, vaginal, rectal, or transdermal),
particularly by inhalation. Parenteral administration includes,
e.g., intravenous, intramuscular, intra-arteriole, intradermal,
subcutaneous, intraperitoneal, intraventricular, and intracranial.
Moreover, where injection is to treat a tumor, e.g., induce
apoptosis, administration may be directly to the tumor and/or into
tissues surrounding the tumor. Other modes of delivery include, but
are not limited to, the use of liposomal formulations, intravenous
infusion, transdermal patches, etc.
[0056] The term "ameliorating" or "ameliorate" refers to any
indicia of success in the treatment of a pathology or condition,
including any objective or subjective parameter such as abatement,
remission or diminishing of symptoms or an improvement in a
patient's physical or mental well-being. Amelioration of symptoms
can be based on objective or subjective parameters; including the
results of a physical examination and/or a psychiatric
evaluation.
[0057] The term "therapy" refers to "treating" or "treatment" of a
disease or condition including preventing the disease or condition
from occurring in a subject (e.g., human) that may be predisposed
to the disease but does not yet experience or exhibit symptoms of
the disease (prophylactic treatment), inhibiting the disease
(slowing or arresting its development), providing relief from the
symptoms or side-effects of the disease (including palliative
treatment), and relieving the disease (causing regression of the
disease).
[0058] The term "effective amount" or "an amount effective to" or a
"therapeutically effective amount" or any gramatically equivalent
term means the amount that, when administered to an animal or human
for treating a disease, is sufficient to effect treatment for that
disease.
[0059] The term "insect cell culture" refers to the in vitro growth
and culturing of cell derived from organisms of the Class Insecta.
"Insect cell culture" also refers to a cell culture comprising
cells of the Class Insecta which have been grown and cultured in
vitro.
[0060] "Peptide" refers to a polymer in which the monomers are
amino acids and are joined together through amide bonds,
alternatively referred to as a polypeptide. Additionally, unnatural
amino acids, for example, .beta.-alanine, phenylglycine and
homoarginine are also included. Amino acids that are not
gene-encoded may also be used in the present invention.
Furthermore, amino acids that have been modified to include
reactive groups, glycosylation sites, polymers, therapeutic
moieties, biomolecules and the like may also be used in the
invention. All of the amino acids used in the present invention may
be either the D- or L-isomer. The L-isomer is generally preferred.
In addition, other peptidomimetics are also useful in the present
invention. As used herein, "peptide" refers to both glycosylated
and unglycosylated peptides. Also included are petides that are
incompletely glycosylated by a system that expresses the peptide.
For a general review, see, Spatola, A. F., in CHEMISTRY AND
BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES AND PROTEINS, B. Weinstein,
eds., Marcel Dekker, New York, p. 267 (1983). The term peptide
includes molecules that are commonly referred to as proteins or
polypeptides.
[0061] A "glycopeptide" as the term is used herein refers to a
peptide having at least one carbohydrate moiety covalently linked
thereto. It is understood that a glycopeptide may be a "therapeutic
glycopeptide". The term "glycopeptide" is used interchangeably
herein with the terms "glycopolypeptide" and "glycoprotein."
[0062] The term "peptide conjugate" refers to species of the
invention in which a peptide is conjugated with a modified sugar as
set forth herein.
[0063] As used herein, the term "modified sugar" refers to a
naturally- or non-naturally-occurring carbohydrate that is
enzymatically added onto an amino acid or a glycosyl residue of a
peptide in a process of the invention. The modified sugar is
selected from a number of enzyme substrates including, but not
limited to sugar nucleotides (mono-, di-, and tri-phosphates),
activated sugars (e.g., glycosyl halides, glycosyl mesylates) and
sugars that are neither activated nor nucleotides. The "modified
sugar" is covalently functionalized with a "modifying group."
Useful modifying groups include, but are not limited to, PEG
moieties, therapeutic moieties, diagnostic moieties, biomolecules
and the like. The modifying group is preferably not a naturally
occurring, or an unmodified carbohydrate. The locus of
functionalization with the modifying group is selected such that it
does not prevent the "modified sugar" from being added
enzymatically to a peptide.
[0064] The term "glycoconjugation" as used herein, refers to the
enzymatically mediated conjugation of a modified sugar species to
an amino acid or glycosyl residue of a polypeptide, e.g., an
erythropoietin peptide prepared by the method of the present
invention. A subgenus of "glycoconjugation" is "glyco-PEGylation,"
in which the modifying group of the modified sugar is poly(ethylene
glycol), an alkyl derivative (e.g., m-PEG) or reactive derivative
(e.g., H.sub.2N-PEG, HOOC-PEG) thereof.
[0065] The terms "large-scale" and "industrial-scale" are used
interchangeably and refer to a reaction cycle or process that
produces at least about 250 mg, preferably at least about 500 mg,
and more preferably at least about 1 gram of peptide at the
completion of a single cycle.
[0066] The term, "glycosyl linking group," as used herein refers to
a glycosyl residue to which a modifying group (e.g., PEG moiety,
therapeutic moiety, biomolecule) is covalently attached; the
glycosyl linking group joins the modifying group to the remainder
of the conjugate. In the methods of the invention, the "glycosyl
linking group" becomes covalently attached to a glycosylated or
unglycosylated polypeptide, thereby linking the modifying group to
an amino acid and/or glycosyl residue of the polypeptide. A
"glycosyl linking group" is generally derived from a "modified
sugar" by the enzymatic attachment of the "modified sugar" to an
amino acid and/or glycosyl residue of the polypeptide. The glycosyl
linking group can be a saccharide-derived structure that is
degraded during formation of modifying group-modified sugar
cassette (e.g., oxidation.fwdarw.Schiff base
formation.fwdarw.reduction), or the glycosyl linking group may be
intact. An "intact glycosyl linking group" refers to a linking
group that is derived from a glycosyl moiety in which the
saccharide monomer that links the modifying group and to the
remainder of the conjugate is not degraded, e.g., oxidized, e.g.,
by sodium metaperiodate. "Intact glycosyl linking groups" of the
invention may be derived from a naturally occurring oligosaccharide
by addition of glycosyl unit(s) or removal of one or more glycosyl
unit from a parent saccharide structure. A "glycosyl linking group"
may include a glycosyl-mimetic moiety. For example, the glycosyl
transferase (e.g., sialyl transferase), which is used to add the
modified sugar to a glycosylated polypeptide, exhibits tolerance
for a glycosyl-mimetic substrate (e.g., a modified sugar in which
the sugar moiety is a glycosyl-mimetic moiety--e.g., sialyl-mimetic
moiety). The transfer of the modified glycosyl-mimetic sugar
results in a conjugate having a glycosyl linking group that is a
glycosyl-mimetic moiety.
[0067] The term "glycosyl-mimetic moiety," as used herein refers to
a moiety, which structurally resembles a glycosyl moiety (e.g., a
hexose or a pentose). Examples of "glycosyl-mimetic moiety" include
those moieties, wherein the glycosidic oxygen or the ring oxygen of
a glycosyl moiety, or both, has been replaced with a bond or
another atom (e.g., sulfur), or another moiety, such as a carbon-
(e.g., CH.sub.2), or nitrogen-containing group (e.g., NH). Examples
include substituted or unsubstituted cyclohexyl derivatives, cyclic
thioethers, cyclic secondary amines, moieties including a
thioglycosidic bond, and the like. In one example, the
"glycosyl-mimetic moiety" is transferred in an enzymatically
catalyzed reaction onto an amino acid residue of a polypeptide or a
glycosyl moiety of a glycopeptide. This can, for instance, be
accomplished by activating the "glycosyl-mimetic moiety" with a
leaving group, such as a halogen.
[0068] The term, "polypeptide glycoform" or "glycoform" as used
herein refers to two polypeptide conjugates having the same amino
acid sequence, but having a different glycosylation pattern with
respect to the glycan residues to which the modifying group(s),
e.g., poly(alkylene oxide) moieties, are covalently linked. Two
polypeptide conjugates having a different number of modifying
groups (e.g., poly(alkylene oxide) moieties) also referred to as
glycoforms. FIG. 3B shows exemplary glycoforms of an EPO
polypeptide conjugate. Illustrated are tri-PEGylated, di-PEGylated
and tetra-PEGylated glycoforms of an EPO-PEG conjugate, wherein the
EPO includes an insect-specific glycosylation pattern. Other
exemplary EPO-PEG glycoforms may be derived from EPO expressed in
CHO cells, as depicted in FIG. 5. Additional EPO-PEG glycoforms,
which may be isolated according to the methods of the invention are
disclosed in U.S. patent application Ser. No. 10/997,405 filed Nov.
24, 2004 and U.S. patent application Ser. No. 11/144,223 filed Jun.
2, 2005, the disclosures of which are disclosed herein in their
entirety. EPO-PEG conjugates discussed in the Examples, below, are
alternatively referred to as EPO-PEG "species", "forms" or
"states".
[0069] The term "isolated" refers to a material that is essentially
free from components, which are used to produce the material. For
peptide conjugates of the invention, the term "isolated" refers to
a material that is essentially free from components which normally
accompany the material in the mixture used to prepare the peptide
conjugate. The terms "isolated" and "pure" are used
interchangeably. Typically, isolated peptide conjugates of the
invention have a level of purity expressed as a range. For example,
the lower end of the range is about 50%, about 55%, about 60%,
about 65%, about 70%, about 75% or about 80% and the upper end of
the range is about 55%, about 60%, about 65%, about 70%, about 75%,
about 80%, about 85%, about 90%, about 95% or more than about
95%.
[0070] When the peptide conjugates are more than about 90% pure,
their purities are preferably expressed as a range. For example,
the lower end of the range being about 90%, about 92%, about 94%,
about 96% or about 98% and the upper end of the range being about
92%, about 94%, about 96%, about 98% or about 100%.
[0071] Methods for the determination of purity are known to those
of skill in the art. Purity of a polypeptide conjugate may be
determined by any suitable, art-recognized method of analysis
(e.g., band intensity on a silver stained gel, polyacrylamide gel
electrophoresis, ELISA, HPLC and the like). An exemplary method is
size-exclusion chromatography (SEC) HPLC, described herein below.
Purity may be determined using relative "area under the curve"
(AUC) values, which can typically be obtained for peaks in a
chromatogram, such as an HPLC chromatogram. Optionally, purities
are determined by chromatographic or other means using a standard
curve generated using a reference material of known purity. Purity
may also be determined on a weight-by-weight basis.
[0072] Methods that are useful for the determination of "purity"
(e.g., those described above) are also useful for the determination
of the "concentration" of a particular component in a mixture
(e.g., a composition of the invention) or relative concentration of
one component with respect to one or more other components. For
example, SEC HPLC may be used to determine the ratio between
different glycoforms or to determine the concentration of a
specific glycoform in a composition of the invention.
[0073] "Essentially each member of the population" as used herein,
speaks to the "homogeneity" of the sites on the peptide and to a
population of peptide that share a common structure, e.g., a common
glycosylation pattern glycosyl structure.
[0074] "Homogeneity" refers to the structural consistency across a
population of polypeptides. Thus, in a glycopeptide of the
invention, in which each glycan residue has the same structure, the
glycopeptide is said to be about 100% homogeneous. Similarly, when
a in a population of glycopeptides, each glycopeptide has glycan
residues of the same structure, such that each peptide of the
population is essentially of the same molecular species, the
population is said to be about 100% homogeneous. Homogeneity is
typically expressed as a range. The lower end of the range of
homogeneity for the peptide conjugates is about 60%, about 70% or
about 80% and the upper end of the range of purity is about 70%,
about 80%, about 90% or more than about 90%.
[0075] When the peptide conjugates are more than or equal to about
90% homogeneous, their homogeneity is also preferably expressed as
a range. The lower end of the range of homogeneity is about 90%,
about 92%, about 94%, about 96% or about 98%. The upper end of the
range of purity is about 92%, about 94%, about 96%, about 98% or
about 100%. The homogeneity of the peptide conjugates is typically
determined by one or more methods known to those of skill in the
art, e.g., gel electrophoresis, liquid chromatography-mass
spectrometry (LC-MS), matrix assisted laser desorption mass time of
flight spectrometry (MALDI-TOF), capillary electrophoresis, and the
like.
[0076] "Substantially uniform glycosylation pattern," when
referring to a glycopeptide species of the invention, refers to the
percentage of glycosylation sites on the polypeptide that have a
glycan residue of the same structure. For example a polypeptide
that includes multiple N-linked or O-linked glycosylation sites may
have a glycosyl residue of the same structure present at all
comparable glycosylation sites, at about 90% of all comparable
sites, about 80% or about 75% of all comparable glycosylation
sites. In these instances the polypeptide would be said to have a
"substantially uniform glycosylation pattern". Alternatively, when
a population of glycopeptides share a common glycosylation pattern,
the population may be said to have a "substantially uniform
glycosylation pattern" when a majority of the peptides in the
population represent essentially a single molecular species.
[0077] For instance, when a population of glycosylated polypeptides
are isolated from a cell, without further modification, the members
of the population may include a range of variations in the precise
structure of their glycan residues. However, in an exemplary
embodiment, peptides isolated from insect cells have a
substantially uniform insect-specific glycosylation pattern. This
refers to the fact that the majority of polypeptides, or
substantially all of the polypeptides, in the preparation represent
one distinct molecular species.
[0078] The term "substantially" in the above definitions of
"substantially uniform" generally means at least about 40%, at
least about 45%, at least about 50%, at least about 55%, at least
about 60%, at least about 65%, at least about 70%, 71%, 72%, 73%,
74%, 75%, 76%, 77%, 78%, 79%, at least about 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or even 100% of the acceptor moieties
are glycosylated with the expected insect cell specific
glycosylation pattern.
[0079] The term "insect specific glycosylation pattern" refers to
the glycosylation pattern found on mature glycopeptides produced by
insect cells. Typically, insect cells generate simple N-linked
oligosaccharides terminating in mannose (for review, see e.g.,
Essentials of Glycobiology A. Varki et al. eds, CSHL Press (1999)
pgs: 32-33). Typically, N-linked glycans produced by insect cell
lines produce glycoproteins that (at maturity) include a
Man.sub.3GlcNAc.sub.2 structure. Fucose units may also be found on
the GlcNAc residue that is directly linked to the peptide. A mature
peptide emerging from a cell with an "insect specific glycosylation
pattern" thus includes one or more glycans having a trimannosyl
(Man.sub.3) or Man.sub.3GlcNAc.sub.2 structure. "Insect specific
glycosylation pattern" also refers to polypeptide populations, in
which essentially all of the polypeptides have glycan structures
terminating with a common motive (e.g., the Man.sub.3 or
Man.sub.3GlcNAc.sub.2 motive) and are not degraded, e.g., to expose
one of the two GlcNAc residues directly bound to the
polypeptide.
[0080] The term "loading buffer" refers to the buffer, in which the
polypeptide conjugate being purified is applied to a purification
device, e.g. a chromatography column or a filter cartridge.
Typically, the loading buffer is selected so that separation of the
peptide conjugate of interest from unwanted impurities can be
accomplished. For instance, when purifying the polypeptide
conjugate on a hydroxyapatite (HA) or fluoroapatite column the pH
of the loading buffer and the salt concentration in the loading
buffer may be selected so that the polypeptide conjugate is
initially retained on the column while certain impurities are found
in the flow through. In other example, the loading buffer is
selected to retain impurities while the desired polypeptide
conjugate is found in the flow-through.
[0081] The term "elution buffer" refers to the buffer, which is
typically used to remove (elute) the polypeptide conjugate from the
purification device (e.g. a chromatographic column or filter
cartridge) to which it was applied earlier. Typically, the elution
buffer is selected so that separation of the polypeptide conjugate
of interest from unwanted impurities can be accomplished. Often,
the concentration of a particular ingredient, such as a particular
salt (e.g. NaCl) in the elution buffer is varied during the elution
procedure (gradient). The gradient may be continuous or stepwise
(interrupted by hold periods).
[0082] The term "controlled room temperature" refers to a
temperature of at least about 10.degree. C., at least about
15.degree. C., at least about 20.degree. C. or at least about
25.degree. C. Typically, controlled room temperature is between
about 20.degree. C. and about 25.degree. C.
I. The Methods
[0083] The present invention provides processes for the isolation
(e.g., large-scale isolation) of polypeptide conjugates from a
mixture. The polypeptide conjugates isolated by the methods of the
invention include at least one modifying group. Exemplary modifying
groups include polymers, such as poly(alkylene oxide) moieties
(e.g., poly(ethylene glycol) or poly(propylene glycol)). Exemplary
modifying groups are described herein, below.
[0084] In one embodiment, the polypeptide conjugate is isolated
from a reaction mixture. In one example, the reaction mixture is
the product of a chemical reaction, such as a chemical PEGylation
reaction. In one example according to this embodiment, the reaction
mixture may contain chemicals, such as unreacted polymeric reagents
and/or hydrolysis products thereof. In another example, the
reaction mixture is the product of an enzymatically catalyzed
reaction, such as an enzymatically catalyzed glycoPEGylation
reaction. In this instance, the reaction mixture may include
enzymes, and may further include reagents, such as unreacted enzyme
substrates (e.g., nucleotide sugars and the like). In one example,
the methods of the invention are suitable for the isolation of a
polypeptide conjugate from the above listed reaction mixture
components.
[0085] In another embodiment, the methods of the invention are
useful to isolate a desired polypeptide conjugate from a mixture
that includes other polypeptide conjugates, which are sought to be
separated from the desired polypeptide conjugate. Such "unwanted"
polypeptide conjugates or side-products may be generated during the
same reaction that leads to the formation of the desired
polypeptide conjugate. For example, a recombinant polypeptide is
subjected to a chemical PEGylation reaction. The reaction product
includes different polypeptide conjugates, in which each type of
polypeptide conjugate includes a different number of PEG moieties,
e.g., the majority of the polypeptide conjugates includes three PEG
moieties, while a small percentage of the polypeptide conjugates in
the reaction mixture is covalently linked to only one or two PEG
moieties. In another example, a recombinantly produced polypeptide
is subjected to an enzymatically catalyzed glycoPEGylation
reaction. Due to a heterogenous glycosylation pattern of the
polypeptide population used in the reaction, the reaction mixture
includes different polypeptide conjugates, in which each type of
polypeptide conjugate has a different structure with respect to the
number of PEG moieties covalently linked to the polypeptide and/or
the structure of the glycan residues, to which each PEG moiety is
attached to the polypeptide. The mixture may also contain unreacted
polypeptide.
[0086] The inventors have discovered that hydrophobic interaction
chromatography (HIC) resins, such as butyl and phenyl resins (e.g.,
Phenyl 650S) are particularly useful for the isolation of
polypeptides modified with at least one poly(alkylene oxide)
moiety. In particular, it has become apparent that HIC is superior
in separating different glycoforms of a polypeptide conjugate,
especially glycoforms that are distinguished by the number of
poly(alkylene oxide) moieties that are linked to the
polypeptide.
[0087] For example, HIC is efficient in separating an
erythropoietin (EPO) conjugate that includes three poly(ethylene
glycol) (PEG) moieties from other EPO conjugates that include 0, 1,
2, 4, 5, 6 or 7 PEG moieties. In addition, HIC can be used to
separate polypeptide conjugates that include the same number of
poly(alkylene oxide) moieties, but wherein the polypeptide
conjugates have a different glycosylation pattern.
[0088] In addition to hydrophobic interaction chromatography, the
methods of the invention may further employ additional
chromatographic steps. In one embodiment, the method includes anion
exchange or mixed-mode chromatography in addition to HIC. In
another embodiment, the method includes cation exchange
chromatography in addition to HIC. In yet another embodiment, the
method includes both, anion exchange or mixed-mode chromatography
and cation exchange chromatography in addition to HIC. In a further
embodiment, the method includes hydroxyapatite or fluoroapatite
chromatography in addition to HIC. The chromatographic steps
employed in the methods of the invention can be performed in any
desired order. In one embodiment, anion exchange or mixed-mode
chromatography is performed prior to hydrophobic interaction
chromatography. In another embodiment, anion exchange or mixed-mode
chromatography is performed after hydrophobic interaction
chromatography. In yet another embodiment, cation exchange
chromatography is performed prior to hydrophobic interaction
chromatography. In a further embodiment, cation exchange
chromatography is performed after hydrophobic interaction
chromatography. In one embodiment, hydroxyapatite or fluoroapatite
chromatography is performed prior to HIC. In another embodiment,
hydroxyapatite or fluoroapatite chromatography is performed after
HIC.
[0089] Hence, in a first aspect, the invention provides a method of
making a composition that includes a first polypeptide conjugate,
wherein the first polypeptide conjugate includes a first number of
poly(alkylene oxide) moieties covalently linked to a first
polypeptide. The method includes: (a) contacting a mixture
containing the first polypeptide conjugate with a hydrophobic
interaction chromatography (HIC) medium; and (b) eluting the first
polypeptide conjugate from the HIC medium. The method may further
include: (c) eluting the first polypeptide conjugate from an anion
exchange or mixed-mode chromatography medium. In one embodiment,
step (c) is performed prior to step (a). In another embodiment,
step (c) is performed after step (b). The method may further
include: (d) eluting the first polypeptide conjugate from a cation
exchange chromatography medium. In one embodiment, step (d) is
performed prior to step (a). In another embodiment, step (d) is
performed after step (b).
[0090] In one embodiment according to this aspect, the mixture
includes additional polypeptide conjugates, from which the first
polypeptide conjugate is isolated. In an exemplary embodiment, the
mixture includes a second polypeptide conjugate, wherein the second
polypeptide conjugate has a second number of poly(alkylene oxide)
moieties covalently linked to a second polypeptide. In one example,
the first polypeptide and the second polypeptide have the same
amino acid sequence. In another example, the first polypeptide and
the second polypeptide have a different amino acid sequence. In one
example, the first number and the second number are different,
which means that the first polypeptide conjugate and the second
polypeptide conjugate are distinguished by the number of
poly(alkylene oxide) moieties that are linked to each polypeptide.
For example, the first polypeptide conjugate includes 3
poly(alkylene oxide) moieties, while the second polypeptide
conjugate includes either 0, 1, 2 or 4 poly(alkylene oxide)
moieties. In one particular example, the first polypeptide and the
second polypeptide have the same amino acid sequence and the first
polypeptide conjugate and the second polypeptide conjugate are
distinguished by a different number of poly(alkylene oxide)
moieties (first number and second number are different).
[0091] In one example, according to any of the above embodiments,
the method of the invention is useful to provide a composition
including a first polypeptide conjugate, wherein the concentration
of the second polypeptide conjugate in this composition is less
than about 30%, less than about 25%, less than about 20%, less than
about 15% and preferably less than about 10%, less than about 9%,
less than about 8%, less than about 7%, less than about 6%, less
than about 5%, less than about 4%, less than about 3%, less than
about 2% or less than about 1%. In another embodiment, the mixture
includes more than one glycoform of the first polypeptide conjugate
and the method provides a composition, in which the combined
concentration of all glycoforms having a structure distinct from
the first polypeptide conjugate is less than about 30%, less than
about 25%, less than about 20%, less than about 15% and preferably
less than about 10%, less than about 9%, less than about 8%, less
than about 7%, less than about 6%, less than about 5%, less than
about 4%, less than about 3%, less than about 2% or less than about
1%.
[0092] In one example according to any of the above embodiments,
the first polypeptide is a glycopeptide and comprises a first
glycosylation pattern that includes at least one glycan residue
covalently linked to the first polypeptide. Each glycan residue can
be linked to at least one polymeric modifying group, such as a
poly(alkylene oxide) moiety. In another example according to the
above embodiments, the first polypeptide includes a first number of
poly(alkylene oxide) moieties, each of which is covalently linked
to the first polypeptide via an N-linked or O-linked glycan.
[0093] In yet another embodiment, the method of the invention is
useful to separate two polypeptide glycoforms that may include the
same number of modifying groups, but that have different
glycosylation patterns. Hence, in one example, the mixture from
which the first polypeptide is isolated, includes a third
polypeptide conjugate that includes a third number of poly(alkylene
oxide) moieties. In one example, the third polypeptide conjugate
and the first polypeptide conjugate include the same number of
poly(alkylene oxide) moieties, but the third polypeptide has a
glycosylation pattern that differs from the glycosylation pattern
of the first polypeptide conjugate by at least one glycosyl moiety.
For example, the third polypeptide conjugate includes a glycan
residue that is not present in the first polypeptide conjugate. In
an exemplary embodiment, the third polypeptide includes an O-linked
glycan, while the first polypeptide includes only N-linked glycans
(see, e.g., FIG. 3B, tri-PEGylated EPO structures). In another
exemplary embodiment, the third polypeptide includes a truncated
glycan residue, while the corresponding glycan residue of the first
polypeptide conjugate is intact (i.e., includes a larger number of
glycosyl moieties).
[0094] In a second aspect, the invention provides a method of
isolating a first polypeptide conjugate including a first number of
poly(alkylene oxide) moieties covalently linked to a first
polypeptide, from a second polypeptide conjugate that includes a
second number of poly(alkylene oxide) moieties covalently linked to
a second polypeptide. The method includes: (a) contacting a mixture
containing the first polypeptide conjugate and the second
polypeptide conjugate with a hydrophobic interaction chromatography
(HIC) medium; and (b) eluting the first polypeptide conjugate from
said hydrophobic interaction chromatography medium. The method may
further include: (c) eluting the first polypeptide conjugate from
an anion exchange chromatography medium. In one embodiment step (c)
is performed prior to step (a). In another embodiment, step (c) is
performed after step (b). The method may further include: (d)
eluting the first polypeptide conjugate from a cation exchange
chromatography medium. In one embodiment, step (d) is performed
prior to step (a). In another embodiment, step (d) is performed
after step (b).
[0095] In one example according to any of the above embodiments,
the first number of poly(alkylene oxide) moieties that are linked
to the first polypeptide is selected from 1 to about 40. In another
example, the first number is selected from 1 to about 30. In yet
another example, the first number is selected from 1 to about 20.
In a further example, the first number is selected from 1, 2, 3, 4,
5, 6, 7, 8, 9 and about 10. In another example, the first
polypeptide conjugate includes exactly three poly(alkylene oxide)
moieties.
[0096] In one example according to any of the above embodiments,
the second number of poly(alkylene oxide) moieties that are linked
to the second polypeptide is selected from 0 to about 40. In
another example, the second number is selected from 0 to about 30.
In yet another example, the second number is selected from 0 to
about 20. In a further example, the second number is selected from
0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and about 10. In another example, the
first number and the second number are different. For example, the
first polypeptide conjugate includes 3 poly(alkylene oxide)
moieties, while the second polypeptide conjugate includes either 0,
1, 2 or 4 poly(alkylene oxide) moieties. One of skill will
understand that a polypeptide that is not linked to a poly(alkylene
oxide) moiety (second number=0), represents unreacted polypeptide
and is not technically a conjugate unless it contains other
modifying groups.
[0097] In one example according to any of the above embodiments,
the first polypeptide and the second polypeptide have the same
amino acid sequence. In another example according to any of the
above embodiments, the first polypeptide is a therapeutic
polypeptide. Exemplary therapeutic polypeptides are described
herein, below. In yet another example according to any of the above
embodiments, the first polypeptide is EPO. In another example
according to any of the above embodiments, both the first
polypeptide and the second polypeptide are EPO. In yet another
example, both the first polypeptide and the third polypeptide are
EPO.
[0098] In one embodiment of the invention, the first polypeptide
conjugate is formed by an enzymatically catalyzed glycomodification
reaction, during which a modified glycosyl moiety [e.g., a glycosyl
moiety modified with at least one poly(alkylene oxide) moiety] is
covalently linked to the first polypeptide. Hence, in one example
according to any of the above embodiments, the method of the
invention may further include: contacting the first polypeptide and
a modified glycosyl donor species (e.g., a modified sugar
nucleotide) having a glycosyl moiety covalently linked to a polymer
(e.g., a poly(alkylene oxide) moiety), in the presence of an enzyme
(e.g., a glycosyltransferase), for which the modified glycosyl
donor species is a substrate, under conditions sufficient for the
enzyme to catalyze the formation of a covalent bond between the
glycosyl moiety that is linked to the polymer and the first
polypeptide. In one example, the modified glycosyl moiety is a
sialic acid (SA) moiety. In one example, the enzyme is a
sialyltransferase. In another example, the polymer is PEG (e.g.,
m-PEG). GlycoPEGylation methods are art-recognized; see for
example, WO 03/031464 to DeFrees et al. or WO 04/99231, the
disclosures of which are incorporated herein by reference in their
entirety.
[0099] In one example according to any of the above embodiments,
the method of the invention may further include: recombinantly
expressing the first polypeptide in a host cell, such as an insect
cell, a mammalian cell (e.g., a CHO cell) or a fungal cell (e.g.,
yeast cell). In one example, the first polypeptide is expressed in
an insect cell line (e.g., a Spodoptera frugiperda cell, e.g.,
Sf9).
[0100] The first polypeptide may be further modified (e.g., through
glycan remodeling) to include a substantially uniform (e.g.,
insect-specific) glycosylation pattern. The glycosylation pattern
of the peptides can be elaborated, trimmed back or otherwise
modified by methods utilizing enzymes. The methods of remodeling
peptides using enzymes that transfer a sugar donor to an acceptor
are discussed in detail in WO 03/031464 to De Frees et al.
(published Apr. 17, 2003); U.S. Patent Application 20040137557
(filed Nov. 5, 2002); U.S. Patent Application 20050143292 (filed
Nov. 24, 2004) and WO 05/051327 (filed Nov. 24, 2004), each of
which is incorporated herein by reference in its entirety.
[0101] Hence, in one embodiment, the method of the invention may
further include: contacting the first polypeptide and a glycosyl
donor molecule (e.g., a nucleotide sugar) in the presence of an
enzyme for which the glycosyl donor molecule is a substrate, under
conditions sufficient for the enzyme to form a covalent bond
between a glycosyl moiety of the glycosyl donor molecule and the
first polypeptide. The polypeptide used as a substrate in this
reaction may be glycosylated or non-glycosylated. The enzyme may be
a glycosyltransferase, such as a GlcNAc-transferase, a
GalNAc-transferase, a Gal-transferase or a sialyltransferase.
[0102] Thus, in one example, the method of the invention includes:
contacting a glycosylated or non-glycosylated first polypeptide and
a nucleotide-N-acetylglucosamine (GlcNAc) or a
nucleotide-N-acetylgalactosamine (GalNAc) molecule in the presence
of a N-acetylglucosamine transferase (e.g., GnT1 or GnT2) or a
N-acetylgalactosamine transferase, respectively. The reaction
mixture may further include a nucleotide galactose (Gal) molecule,
and a galactosyl transferase (e.g., GalT1). The components of the
reaction mixture are contacted (e.g., in a single reaction vessel
or sequentially) under conditions sufficient for the
N-acetylglucosamine transferase and the galactosyl transferase to
form a glycosylated first polypeptide having at least one glycan
residue with a terminal -GlcNAc-Gal moiety or a GalNAc-Gal moiety.
That glycan residue is preferably mono-antennary with respect to
the newly added GlcNAc-Gal or -GalNAc-Gal moiety. In one
embodiment, the GlcNAc-Gal moiety is added to a mannose residue,
which is part of a tri-mannosyl motif. In another embodiment, the
-GalNAc-Gal moiety is added to a serine or threonine residue of the
first polypeptide.
[0103] In a third aspect, the invention provides a method of making
a composition that contains a first erythropoietin (EPO) conjugate,
wherein the first EPO conjugate includes a first number of
poly(alkylene oxide) moieties covalently linked to an EPO
polypeptide. The method includes: (a) contacting a mixture
containing the first EPO conjugate with an anion exchange medium;
(b) eluting the first EPO conjugate from the anion exchange medium,
forming a first eluate including the first EPO conjugate; (c)
contacting the first eluate with a hydrophobic interaction
chromatography (HIC) medium; and (d) eluting the first EPO
conjugate from the hydrophobic interaction chromatography medium.
The method may further include: (e) eluting the first EPO conjugate
from a cation exchange chromatography medium. In one example, step
(e) is performed after step (d). In another example, step (e) is
performed prior to step (c). The method may further include one or
more dilution or diafiltration steps. In one example, diafiltration
is used to concentrate and/or exchange the buffer in order to
condition the sample for the next process step. For example, the
eluate from the HIC step is concentrated and diafiltered into a new
buffer system in order to prepare the sample for cation exchange
chromatography.
[0104] In one embodiment according to this aspect, the mixture
includes additional EPO conjugates, from which the first EPO
conjugate is isolated. In an exemplary embodiment, the mixture
includes a second EPO conjugate having a second number of
poly(alkylene oxide) moieties covalently linked to an EPO
polypeptide. In one embodiment, the first number and the second
number are different, which means that the first EPO conjugate and
the second EPO conjugate are glycoforms distinguished by the number
of poly(alkylene oxide) moieties that are linked to each EPO
polypeptide. For example, the first EPO conjugate includes 3
poly(alkylene oxide) moieties, while the second EPO conjugate may
include 0, 1, 2 or 4 poly(alkylene oxide) moieties. In one example,
the method is useful to provide a composition including a first EPO
conjugate, wherein the concentration of the second EPO conjugate in
this composition is less than about 30%, less than about 25%, less
than about 20%, less than about 15% and preferably less than about
10%, less than about 9%, less than about 8%, less than about 7%,
less than about 6%, less than about 5%, less than about 4%, less
than about 3%, less than about 2% or less than about 1%. In another
embodiment, the mixture includes more than one glycoform of the
first EPO conjugate and the method provides a composition, in which
the combined concentration of all glycoforms having a structure
distinct from the structure of the first EPO conjugate is less than
about 30%, less than about 25%, less than about 20%, less than
about 15% and preferably less than about 10%, less than about 9%,
less than about 8%, less than about 7%, less than about 6%, less
than about 5%, less than about 4%, less than about 3%, less than
about 2% or less than about 1%.
[0105] An exemplary EPO sequence useful in conjunction with any of
the above embodiments, is represented by SEQ ID NO:1, which may
include at least one mutation (e.g., Arg.sup.139 to Ala.sup.139,
Arg.sup.143 to Ala.sup.143 and Lys.sup.154 to Ala.sup.154). In
another exemplary embodiment, the EPO conjugates of the invention
may include at least one N-linked glycan residue. In one example,
the N-linked glycan residue is covalently linked to an amino acid
residue selected from Asn.sup.24, Asn.sup.38 and Asn.sup.83 of SEQ
ID NO:1. The EPO conjugate may further include an O-linked glycan
residue. In one example, the O-linked glycan residue is covalently
linked to a serine (e.g., Ser 126) residue of SEQ ID NO:1. Any of
the above described glycan residues can optionally be linked to a
poly(alkylene oxide) moiety. In one example according to any of the
above embodiments, EPO is covalently linked to three poly(alkylene
oxide) moieties (e.g., three PEG moieties). In another example
according to any of the above embodiments, EPO is covalently linked
to three poly(alkylene oxide) moieties (e.g., PEG), wherein at
least two of the three poly(alkylene oxide) moieties are covalently
linked to the EPO polypeptide via N-linked glycans. In one example,
at least one N-linked glycan is mono-antennary. In another example,
all three N-linked glycans are mono-antennary (e.g., as shown in
FIG. 3A). Exemplary tri-PEGylated EPO conjugates are shown in FIG.
3B.
[0106] In one embodiment, the EPO conjugate is formed by an
enzymatically catalyzed glycomodification reaction, wherein a
modified glycosyl moiety (e.g., a glycosyl moiety modified with at
least one poly(alkylene oxide) moiety) is attached to the EPO
polypeptide. Hence, in one example according to any of the above
embodiments, the method of the invention may further include:
contacting an EPO polypeptide and a modified glycosyl donor species
(e.g., a modified sugar nucleotide) having a glycosyl moiety
covalently linked to a polymer (e.g., a poly(alkylene oxide)
moiety), in the presence of an enzyme (e.g., a
glycosyltransferase), for which the modified glycosyl donor species
is a substrate, under conditions sufficient for the enzyme to
catalyze the formation of a covalent bond between the glycosyl
moiety that is linked to the polymer and the EPO polypeptide. In
one example, the modified glycosyl moiety is a sialic acid (SA)
moiety. In another example, the enzyme is a sialyltransferase. The
method may further include: recombinantly expressing the EPO
polypeptide in a host cell, such as a bacterial (e.g., E. coli), an
insect cell, a mammalian cell (e.g., CHO) cell or a fungal cell. In
one example, the EPO polypeptide is expressed in an insect cell
line (e.g., Sf9) and is optionally purified from insect cell
culture, e.g., according to the methods outlined in WO 06/105426 to
Kang et al.
[0107] The EPO peptide may be further modified through glycan
remodeling to include a substantially uniform (e.g.,
insect-specific) glycosylation pattern. Hence, in one embodiment,
the method of the invention may further include: contacting (e.g.,
in a single reaction vessel) a glycosylated EPO polypeptide with a
nucleotide-N-acetylglucosamine (GlcNAc) molecule and a nucleotide
galactose (Gal) molecule in the presence of a N-acetylglucosamine
transferase (e.g., GnT1 or GnT2), and a galactosyl transferase
(e.g., GalT1), under conditions sufficient for said
N-acetylglucosamine transferase and said galactosyl transferase to
form a glycosylated EPO polypeptide having at least one glycan
residue with a terminal -GlcNAc-Gal moiety. That glycan residue is
preferably mono-antennary with respect to the newly added
GlcNAc-Gal moiety. In one embodiment, the -GlcNAc-Gal moiety is
added to a mannose residue, which is part of a tri-mannosyl
motive.
[0108] In one example according to any of the above embodiments,
each poly(alkylene oxide) moiety is a member independently selected
from poly(ethylene glycol) (e.g., m-PEG) and poly(propylene glycol)
(e.g., m-PPG). Exemplary poly(ethylene glycol) moieties are
described herein, below. In another example according to any of the
above embodiments, each poly(alkylene oxide) moiety has an
independently selected molecular weight between about 1 kDa and
about 200 kDa. Additional molecular weight ranges for poly(alkylene
oxide) moieties are given herein, below.
[0109] In one example according to any of the above embodiments,
the first polypeptide conjugate includes at least one poly(alkylene
oxide) moiety that is covalently linked to the first polypeptide
via a glycosyl linking group. In one example, the glycosyl linking
group is covalently linked to an amino acid residue of the first
polypeptide. In another example, the glycosyl linking group is
covalently linked to a glycosyl moiety of said first polypeptide.
In yet another example, the glycosyl linking group is an intact
glycosyl linking group. Exemplary glycosyl linking groups are
described herein and, for example, in WO 03/031464 to DeFrees et
al., WO 04/99231, and PCT/U.S.07/74139 filed Jul. 23, 2007, the
disclosures of which are incorporated herein by reference in their
entirety. Exemplary intact glycosyl linking groups include sialic
acid moieties, GlcNH and GlcNAc moieties, as well as Gal, GalNH and
GalNAc moieties.
[0110] Exemplary HIC media that are useful in any of the above
described embodiments, include butyl and phenyl resins, such as
Phenyl 650S (e.g., ToyoPearl). Hydrophobic interaction
chromatography and suitable HIC media are described herein below
and, for example in Process Scale Bioseparations for the
Biopharmaceutical Industry, Ed. Shukla A A, Etzel M R, Gadam S, CRC
Press Taylor & Francis Group (2007), pages 197-206, the
disclosure of which is incorporated herein by reference.
[0111] Those of skill in the art will appreciate that the methods
of the invention can be practiced for polypeptide conjugates based
on a wide variety of polypeptides. The methods are not limited to a
particular polypeptide. Hence, any of the above described
embodiments of the invention can be practiced with any of the below
described polypeptide conjugates.
I.a) Polypeptide Conjugates
[0112] The polypeptide conjugates isolated by the methods of the
invention include a polypeptide and at least one modifying group
covalently linked to the polypeptide, e.g., via a glycosyl linking
group. Exemplary polypeptide conjugates are discussed herein below
and, for example WO 03/031464 to DeFrees et al., WO 04/99231 to
DeFrees et al., and WO 04/33651 to DeFrees et al., the disclosures
of which are incorporated herein by reference in their
entirety.
Polypeptides
[0113] The polypeptide that is part of polypeptide conjugates of
the invention can be any glycosylated or non-glycosylated
polypeptide. In one embodiment, the polypeptide is a recombinant
polypeptide. In one example according to this embodiment, the
polypeptide is expressed in a host cell selected from bacterial
cells (e.g., E. coli), insect cells (e.g., Spodoptera frugiperda
cells), fungal cells (e.g., yeast cells), mammalian cells (e.g.,
CHO cells) and bacterial cells (e.g., E. coli cells). Methods for
the expression of polypeptides in insect cell lines are discussed
herein below. In another embodiment, the polypeptide is chemically
synthesized and optionally includes non-natural amino acids.
[0114] The polypeptide can have any number of amino acids. In one
embodiment, the peptide or glycopeptide has a molecular weight of
about 5 kDa to about 500 kDa. In another embodiment, the peptide or
glycopeptide has a molecular weight of about 10 kDa to about 100
kDa. In yet another embodiment, the polypeptide has a molecular
weight of about 10 kDa to about 30 kDa. In a further embodiment,
the polypeptide has a molecular weight of about 20 kDa to about 25
kDa.
[0115] Exemplary polypeptides include wild-type polypeptides and
fragments thereof as well as polypeptides, which are modified from
their naturally occurring counterpart (e.g., by mutation or
truncation). A polypeptide may also be a fusion protein. Exemplary
fusion proteins include those, in which the polypeptide is fused to
a fluorescent protein (e.g., GFP), a therapeutic polypeptide, an
antibody, a receptor ligand, a proteinaceous toxin, MBP, a Histag
and the like.
[0116] In one embodiment, the polypeptide is a therapeutic
polypeptide (i.e., authorized drug), such as those currently used
as pharmaceutical agents. A non-limiting selection of polypeptides
is shown in FIG. 28 of U.S. patent application Ser. No. 10/552,896
filed Jun. 8, 2006, which is incorporated herein by reference.
[0117] Exemplary polypeptides include growth factors, such as
fibroblast growth factors (e.g., FGF-1, FGF-2, FGF-3, FGF-4, FGF-5,
FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14,
FGF-15, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22 and
FGF-23), blood coagulation factors (e.g., Factor V, Factor VII,
Factor VIII, B-domain deleted Factor VIII, partial B-domain deleted
Factor VIII, vWF-Factor VIII fusion (e.g., with full-length or
B-domain deleted Factor VIII), Factor IX, Factor X and Factor
XIII), hormones, such as human growth hormone (hGH) and follicle
stimulating hormone (FSH), as well as cytokines, such as
interleukins (e.g., IL-1, IL-2, IL-12) and interferons (e.g.,
INF-alpha, INF-beta, INF-gamma).
[0118] Other exemplary polypeptides include enzymes, such as
glucocerebrosidase, alpha-galactosidase (e.g., Fabrazyme.TM.),
acid-alpha-glucosidase (acid maltase), alpha-L-iduronidase (e.g.,
Aldurazyme.TM.), thyroid peroxidase (TPO), beta-glucosidase (see
e.g., enzymes described in U.S. patent application Ser. No.
10/411,044), and alpha-galactosidase A (see e.g., enzymes described
in U.S. Pat. No. 7,125,843).
[0119] Other exemplary parent polypeptides include bone
morphogenetic proteins (e.g., BMP-1, BMP-2, BMP-3, BMP-4, BMP-5,
BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14,
BMP-15), neurotrophins (e.g., NT-3, NT-4, NT-5), erythropoietins
(EPO), growth differentiation factors (e.g., GDF-5), glial cell
line-derived neurotrophic factor (GDNF), brain derived neurotrophic
factor (BDNF), nerve growth factor (NGF), von Willebrand factor
(vWF), vWF protease, granulocyte colony stimulating factor (G-CSF),
granulocyte-macrophage colony stimulating factor (GM-CSF),
.alpha..sub.1-antitrypsin (ATT, or .alpha.-1 protease inhibitor),
tissue-type plasminogen activator (TPA), hirudin, leptin,
urokinase, human DNase, insulin, hepatitis B surface protein
(HbsAg), human chorionic gonadotropin (hCG), chimeric diphtheria
toxin-IL-2, glucagon-like peptides (e.g., GLP-1 and GLP-2),
anti-thrombin III (AT-III), prokinetisin, CD4, .alpha.-CD.sub.20,
tumor necrosis factor receptor (TNF-R), P-selectin glycoprotein
ligand-1 (PSGL-1), complement, transferrin, glycosylation-dependent
cell adhesion molecule (GlyCAM), neural-cell adhesion molecule
(N-CAM), TNF receptor-IgG Fc region fusion protein and extendin-4.
Exemplary amino acid sequences for some of the above listed
polypeptides are described in U.S. Pat. No. 7,214,660, all of which
are incorporated herein by reference.
[0120] In an exemplary embodiment, the polypeptide is EPO
comprising the amino acid sequence of (SEQ ID NO:1), which is shown
below:
TABLE-US-00001 Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu
Arg Tyr Leu Leu Glu Ala Lys Glu Ala Glu Ile Thr Thr Gly Cys Ala Glu
His Cys Ser Leu Asn Glu Asn.sup.38 Ile Thr Val Pro Asp Thr Lys Val
Asn Phe Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val
Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu
Leu Val Asn.sup.83 Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val
Asp Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu
Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ala Ala Pro Leu
Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn
Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly
Asp
[0121] Also within the scope of the invention are polypeptides that
are antibodies. The term antibody is meant to include antibody
fragments (e.g., Fc domains), single chain antibodies, Lama
antibodies, nano-bodies and the like. Also included in the term are
antibody-fusion proteins, such as Ig chimeras. Preferred antibodies
include humanized, monoclonal antibodies or fragments thereof. All
known isotypes of such antibodies are within the scope of the
invention. Exemplary antibodies include those to growth factors,
such as endothelial growth factor (EGF), vascular endothelial
growth factors (e.g., monoclonal antibody to VEGF-A, such as
ranibizumab (Lucentis.TM.)) and fibroblast growth factors, such as
FGF-7, FGF-21 and FGF-23) and antibodies to their respective
receptors. Other exemplary antibodies include anti-TNF-alpha
monoclonal antibodies (see e.g., U.S. patent application Ser. No.
10/411,043), TNF receptor-IgG Fc region fusion protein (e.g.,
Enbrel.TM.), anti-HER2 monoclonal antibodies (e.g., Herceptin.TM.),
monoclonal antibodies to protein F of respiratory syncytial virus
(e.g., Synagis.TM.), monoclonal antibodies to TNF-.alpha. (e.g.,
Remicade.TM.), monoclonal antibodies to glycoproteins, such as
IIb/IIIa (e.g., Reopro.TM.), monoclonal antibodies to CD20 (e.g.,
Rituxan.TM.), CD4 and alpha-CD3, monoclonal antibodies to PSGL-1
and CEA. Any modified (e.g., mutated) version of any of the above
listed polypeptides is also within the scope of the invention.
Polypeptides Expressed in Insect Cells
[0122] In one embodiment, the polypeptide is expressed in insect
cells. Insect cells suitable for use in the present invention are
from any order of the class Insecta which can be hosts to
recombinant viruses (e.g. baculovirus) or wild-type viruses, and
which can grow and produce recombinant peptide products upon
infection with the virus in a medium composition of the invention.
In an exemplary embodiment, the cells are from the Diptera or
Lepidoptera orders. Preferred are insect cell lines that can be
used to produce polypeptides having a substantially uniform,
insect-specific glycosylation pattern. In one embodiment, the
polypeptide is expressed by a stably transfected cell.
[0123] About 300 insect species have been reported to have nuclear
polyhedrosis virus (NPV) diseases, the majority (243) of which were
isolated from Lepidoptera (see e.g., Weiss et al., Cell Culture
Methods for Large-Scale Propagation of Baculoviruses, In Granados
et al. (eds.), The Biology of Baculoviruses: Vol. II Practical
Application for Insect Control, pp. 63-87 at p. 64 (1986)). Insect
cell lines derived from the following insects are exemplary:
Carpocapsa pomonella (preferably cell line CP-128); Trichoplusia ni
(preferably cell line TN-368); Autographa californica; Spodoptera
frugiperda (preferably cell line Sf9); Lymantria dispar; Mamestra
brassicae; Aedes albopictus; Orgyia pseudotsugata; Neodiprion
sertifer; Aedes aegypti; Antheraea eucalypti; Gnorimoschema
opercullela; Galleria mellonella; Spodoptera littoralis; Drosophila
melanogaster, Heliothis zea; Spodoptera exigua; Rachiplusia ou;
Plodia interpunctella; Amsacta moorei; Agrotis c-nitrum, Adoxophyes
orana, Agrotis segetum, Bombyx mori, Hyponomeuta malinellus, Colias
eurytheme, Anticarsia gemmetalis, Apanteles melanoscelus, Arctia
caja, and Lymantria dispar.
[0124] In an exemplary embodiment, the insect cells are from
Spodoptera frugiperda, and in another exemplary embodiment, the
cell line is Sf9 (ATCC CRL 1711). Sf9, Sf21, and High-Five insect
cells are commonly used for baculovirus expression. Sf9 and Sf21
are ovarian cell lines from Spodoptera frugiperda. High-Five cells
are egg cells from Trichoplusia ni. Sf9, Sf21 and High-Five cell
lines may be grown at room temperature (e.g. 25 to 27.degree. C.),
and do not require CO.sub.2 incubators. Their doubling time is
between about 18 and 24 hours. The insect cell lines cultured to
produce the peptides and glycopeptides of the invention are
preferably those suitable for the reproduction of numerous
insect-pathogenic viruses such as picornaviruses, parvoviruses,
entomopox viruses, baculoviruses and rhabdoviruses. In an exemplary
embodiment, nucleopolyhedrosis viruses (NPV) and granulosis viruses
(GV) from the group of baculoviruses are preferred.
[0125] Baculoviruses are characterized by rod-shaped virus
particles which are generally occluded in occlusion bodies (also
called polyhedra). The family Baculoviridae can be divided in two
subfamilies: the Eubaculovirinae comprising two genera of occluded
viruses; nuclear polyhedrosis virus (NPV) and granulosis virus
(GV), and the subfamily Nudobaculovirinae comprising the
nonoccluded viruses.
[0126] Methods of preparing and using virus expression systems are
generally known in the art. For example, with respect to
baculovirus systems, representative references include U.S. Pat.
No. 5,194,376, U.S. Pat. No. 5,147,788, U.S. Pat. No. 4,879,236 and
Bedard C. et al. (1994) Cytotechnology 15:129-138; Hink W T et al.,
(1991) Biotechnology Progress 7:9-14; Licari P. et al., (1992)
Biotechnology and Bioengineering 39:614-618, each of which is
incorporated herein by reference in its entirety. The incorporation
of a desired nucleic acid into a baculovirus expression vector may
be accomplished using techniques that are well known in the art.
For example, such techniques are described in, Sambrook et al.
(Third Edition, 2001, Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York), and in Ausubel et al. (1997),
Current Protocols in Molecular Biology, John Wiley & Sons, New
York).
[0127] In one embodiment, the polypeptide expressed in any suitable
expression system, is isolated from cell culture before the
polypeptide is modified with a modifying group. In one example, the
polypeptide is first removed from the cell culture medium, cellular
debris and other particles and is then further purified to remove
contaminants, such as viral particles and unwanted proteins, using
a variety of filtration and chromatographic purification devices.
Polypeptide purification techniques are known. See, e.g., Protein
Purification Methods, A Practical Approach, Ed. Harris E L V, Angal
S, IRL Press Oxford, England (1989), Protein Purification, Ed.
Janson J C, Ryden L, VCH-Verlag, Weinheim, Germany (1989), Process
Scale Bioseparations for the Biopharmaceutical Industry, Ed. Shukla
A A, Etzel M R, Gadam S, CRC Press Taylor & Francis Group
(2007), and Protein Purification, Principles, High Resolution
Methods and Applications (2.sup.nd Edition 1998), Ed. Janson J-C
and Ryden L. Exemplary methods for the isolation of polypeptides
expressed in insect cells are also disclosed in WO 06/105426 to
Kang et al.
Modifying Group
[0128] The modifying group of the invention can be any chemical
moiety. Exemplary modifying groups are discussed below.
Polymeric Modifying Groups
[0129] In one embodiment, the modifying group is a linear or
branched polymeric modifying group (polymer). A polymeric modifying
group includes at least one polymeric moiety, wherein each
polymeric moiety is independently selected. In another embodiment,
the polymeric modifying group is water-soluble. A water-soluble
polymeric modifying group includes at least one polar group.
Exemplary polar groups, include polyether groups, hydroxyl groups
and carboxylic acid groups.
[0130] Many water-soluble polymers are known to those of skill in
the art and are useful in practicing the present invention. The
term water-soluble polymer encompasses species such as saccharides
(e.g., dextran, amylose, hyalouronic acid, poly(sialic acid),
heparans, heparins, etc.); poly(amino acids), e.g., poly(aspartic
acid) and poly(glutamic acid); nucleic acids; synthetic polymers
(e.g., poly(acrylic acid), poly(alkylene oxides), peptides,
proteins, and the like. In a preferred embodiment, the polymer is a
poly(alkylene oxide), such as a poly(ethylene glycol) or a
polypropylene glycol.
[0131] In one example, the water-soluble polymer is polyethylene
glycol (PEG) or a PEG analog, e.g., methoxy-poly(ethylene glycol)
(m-PEG). In another example, the water-soluble polymer is
polypropylene glycol (PPG), e.g., methoxy-polypropylene glycol
(m-PPG). PEG is frequently used to modify the properties of
polypeptides, such as therapeutic proteins. For example, the in
vivo half-life of therapeutic glycopeptides can be enhanced with
PEG moieties. Chemical modification of polypeptides with PEG
(PEGylation) increases their molecular size and typically decreases
surface- and functional group-accessibility, each of which are
dependent on the number and size of the PEG moieties attached to
the polypeptide. Frequently, this modification results in an
improvement of plasma half-live and in proteolytic-stability, as
well as a decrease in immunogenicity and hepatic uptake (Chaffee et
al. J. Clin. Invest. 89: 1643-1651 (1992); Pyatak et al. Res.
Commun. Chem. Pathol Pharmacol. 29: 113-127 (1980)). For example,
PEGylation of interleukin-2 has been reported to increase its
antitumor potency in vivo (Katre et al. Proc. Natl. Acad. Sci. USA.
84: 1487-1491 (1987)) and PEGylation of a F(ab')2 derived from the
monoclonal antibody A7 has improved its tumor localization
(Kitamura et al. Biochem. Biophys. Res. Commun. 28: 1387-1394
(1990)). Thus, in another embodiment, the in vivo half-life of a
polypeptide derivatized with a PEG moiety by a method of the
invention is increased relative to the in vivo half-life of the
non-derivatized parent polypeptide.
[0132] The poly(ethylene glycol) or poly(propylene glycol) is not
restricted to any particular form or molecular weight range. The
size of these modifying groups may, for example, depend on the
nature and size of the polypeptide to which they are attached and
the properties desired for the modified polypeptide. For unbranched
poly(ethylene glycol) or poly(propylene glycol) molecules the
molecular weight is preferably between about 0.5 kDa and about 500
kDa. Branched polymers may be larger than 500 kDa. In one
embodiment, branched poly(ethylene glycol) or poly(propylene
glycol) have a molecular weight from about 0.5 kDa to about 1000
kDa.
[0133] In an exemplary embodiment, the PEG or PPG molecule of use
in the invention (branched or unbranched) has a molecular weight
selected from about 0.5 kDa, 1 kDa, about 2 kDa, about 5 kDa, about
10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa,
about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, about 55
kDa, about 60 kDa, about 65 kDa, about 70 kDa, about 75 kDa, about
80 kDa, about 85 kDa, about 90 kDa, about 95 kDa, about 100 kDa,
about 105 kDa, about 110 kDa, about 115 kDa, about 120 kDa, about
125 kDa, about 130 kDa, about 135 kDa, about 140 kDa, about 145
kDa, about 150 kDa, about 155 kDa, about 160 kDa, about 165 kDa,
about 170 kDa, about 175 kDa, about 180 kDa, about 185 kDa, about
190 kDa, about 195 kDa and about 400 kDa.
[0134] In one embodiment, the polypeptide is EPO. In another
embodiment, the EPO peptide has at least two, and preferably three
poly(ethylene glycol) moieties covalently linked thereto. In one
example according to this embodiment, each PEG molecule linked to
the EPO peptide has a molecular weight from about 2 kDa to about 80
kDa, preferably from about 5 kDa to about 60 kDa and more
preferably from about 10 kDa to about 40 kDa.
[0135] Exemplary water-soluble polymers are those in which a
substantial proportion of the polymer molecules in a sample of the
polymer are of approximately the same molecular weight; such
polymers are "homodisperse."
[0136] The following discussion of polymers including PEG moieties
is for clarity of illustration. Those of skill will appreciate that
the focus in the sections that follow and the various motifs set
forth using PEG as an exemplary polymer are equally applicable to
species in which a polymer other than PEG (e.g., another
poly(alkylene oxide)) is utilized.
[0137] Exemplary poly(ethylene glycol) molecules of use in the
invention include, but are not limited to, those having the
formula:
##STR00001##
in which R.sup.8 is H, OH, NH.sub.2, substituted or unsubstituted
alkyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted heteroalkyl, e.g.,
acetal, OHC--, H.sub.2N--(CH.sub.2).sub.q--, HS--(CH.sub.2).sub.q,
or --(CH.sub.2).sub.qC(Y)Z.sup.1. The index "e" represents an
integer from 1 to 2500. The indices b, d, and q independently
represent integers from 0 to 20. The symbols Z and Z.sup.1
independently represent OH, NH.sub.2, leaving groups, e.g.,
imidazole, p-nitrophenyl, HOBT, tetrazole, halide, S--R.sup.9, the
alcohol portion of activated esters; --(CH.sub.2).sub.pC(Y.sup.1)V,
or --(CH.sub.2).sub.pU(CH.sub.2).sub.sC(Y.sup.1).sub.v. The symbol
Y represents H(2), .dbd.O, .dbd.S, .dbd.N--R.sup.10. The symbols X,
Y, Y.sup.1, A.sup.1, and U independently represent the moieties O,
S, N--R''. The symbol V represents OH, NH.sub.2, halogen,
S--R.sup.12, the alcohol component of activated esters, the amine
component of activated amides, sugar-nucleotides, and proteins. The
indices p, q, s and v are members independently selected from the
integers from 0 to 20. The symbols R.sup.9, R.sup.10, R.sup.11 and
R.sup.12 independently represent H, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heterocycloalkyl
and substituted or unsubstituted heteroaryl.
[0138] The poly(ethylene glycol) useful in forming the conjugate of
the invention is either linear or branched. Branched poly(ethylene
glycol) molecules suitable for use in the invention include, but
are not limited to those described by the following formula:
##STR00002##
in which R.sup.8 and R.sup.8' are members independently selected
from the groups defined for R.sup.8, above. A.sup.1 and A.sup.2 are
members independently selected from the groups defined for A.sup.1,
above. The indices e, f, o, and q are as described above. Z and Y
are as described above. X.sup.1 and X.sup.1' are members
independently selected from S, SC(O)NH, HNC(O)S, SC(O)O, O, NH,
NHC(O), (O)CNH and NHC(O)O, OC(O)NH.
[0139] In other exemplary embodiments, the branched PEG is based
upon a cysteine, serine or di-lysine core. In another exemplary
embodiments, the poly(ethylene glycol) molecule is selected from
the following structures:
##STR00003##
[0140] In a further embodiment the poly(ethylene glycol) is a
branched PEG having more than one PEG moiety attached. Examples of
branched PEGs are described in U.S. Pat. No. 5,932,462; U.S. Pat.
No. 5,342,940; U.S. Pat. No. 5,643,575; U.S. Pat. No. 5,919,455;
U.S. Pat. No. 6,113,906; U.S. Pat. No. 5,183,660; WO 02/09766;
Kodera Y., Bioconjugate Chemistry 5: 283-288 (1994); and Yamasaki
et al., Agric. Biol. Chem., 52: 2125-2127, 1998. In a preferred
embodiment the molecular weight of each poly(ethylene glycol) of
the branched PEG is less than or equal to 40,000 daltons.
[0141] Representative polymeric modifying moieties include
structures that are based on side chain-containing amino acids,
e.g., serine, cysteine, lysine, and small peptides, e.g., lys-lys.
Exemplary structures include:
##STR00004##
[0142] Those of skill will appreciate that the free amine in the
di-lysine structures can also be pegylated through an amide or
urethane bond with a PEG moiety.
[0143] In yet another embodiment, the polymeric modifying moiety is
a branched PEG moiety that is based upon a tri-lysine peptide. The
tri-lysine can be mono-, di-, tri-, or tetra-PEG-ylated. Exemplary
species according to this embodiment have the formulae:
##STR00005##
in which the indices e, f and f' are independently selected
integers from 1 to 2500; and the indices q, q' and q'' are
independently selected integers from 1 to 20.
[0144] As will be apparent to those of skill, the branched polymers
of use in the invention include variations on the themes set forth
above. For example the di-lysine-PEG conjugate shown above can
include three polymeric subunits, the third bonded to the
.alpha.-amine shown as unmodified in the structure above.
Similarly, the use of a tri-lysine functionalized with three or
four polymeric subunits labeled with the polymeric modifying moiety
in a desired manner is within the scope of the invention.
[0145] An exemplary branched modifying group including one or more
polymeric moieties (e.g., PEG moieties) includes the formula:
##STR00006##
[0146] In one embodiment, the branched polymer species according to
this formula are essentially pure water-soluble polymers. C is
carbon. X.sup.5 is a non-reactive group. In one embodiment, X.sup.5
is selected from H, OH and C.sub.1-C.sub.6 alkyl (e.g., CH.sub.3,
--CH.sub.2CH.sub.3) optionally substituted with OH. R.sup.16 and
R.sup.17 are independently selected from non-reactive groups (e.g.,
H, unsubstituted alkyl, unsubstituted heteroalkyl) and polymeric
arms (e.g., PEG). X.sup.2 and X.sup.4 are linkage fragments that
are preferably essentially non-reactive under physiological
conditions. X.sup.2 and X.sup.4 are independently selected. An
exemplary linker includes neither aromatic nor ester moieties.
Alternatively, these linkages can include one or more moiety that
is designed to degrade under physiologically relevant conditions,
e.g., esters, disulfides, etc. X.sup.2 and X.sup.4 join the
polymeric arms R.sup.16 and R.sup.17 to C. Exemplary linkage
fragments including X.sup.2 and X.sup.4 are independently selected
and include S, SC(O)NH, HNC(O)S, SC(O)O, O, NH, NHC(O), (O)CNH and
NHC(O)O, and OC(O)NH, CH.sub.2S, CH.sub.2O, CH.sub.2CH.sub.2O,
CH.sub.2CH.sub.2S, (CH.sub.2).sub.oO, (CH.sub.2).sub.oS or
(CH.sub.2).sub.oY'-PEG wherein, Y' is S, NH, NHC(O), C(O)NH,
NHC(O)O, OC(O)NH, or O and o is an integer from 1 to 50. In an
exemplary embodiment, the linkage fragments X.sup.2 and X.sup.4 are
different linkage fragments.
[0147] In an exemplary embodiment, the modifying group is derived
from a natural or unnatural amino acid, amino acid analog or amino
acid mimetic, or a small peptide formed from one or more such
species. For example, certain branched polymers found in the
polypeptide conjugates of the invention have the formula, wherein
La is a linker moiety that links the modifying group to the
remainder of the polypeptide conjugate.
##STR00007##
[0148] In an exemplary embodiment, L.sup.a is a linking moiety
having the structure:
##STR00008##
in which X.sup.a and X.sup.b are independently selected linkage
fragments and L.sup.1 is selected from a bond, substituted or
unsubstituted alkyl or substituted or unsubstituted
heteroalkyl.
[0149] Exemplary species for X.sup.a and X.sup.b include S,
SC(O)NH, HNC(O)S, SC(O)O, O, NH, NHC(O), C(O)NH and NHC(O)O, and
OC(O)NH.
[0150] In another exemplary embodiment, X.sup.4 is a peptide bond
to R.sup.17, which is an amino acid, di-peptide (e.g., Lys-Lys) or
tri-peptide (e.g., Lys-Lys-Lys) in which the alpha-amine
moiety(ies) and/or side chain heteroatom(s) are modified with a
polymeric modifying moiety.
[0151] In other exemplary embodiments, the polypeptide conjugate
includes a moiety selected from the group:
##STR00009##
[0152] In each of the formulae above, the indices e and f are
independently selected from the integers from 1 to 2500. In further
exemplary embodiments, e and f are selected to provide a PEG moiety
that is about 1 kDa, 2 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa,
30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70
kDa, 75 kDa and 80 kDa. The symbol Q represents substituted or
unsubstituted alkyl (e.g., C.sub.1-C.sub.6 alkyl, e.g., methyl),
substituted or unsubstituted heteroalkyl or H.
[0153] Other branched polymers have structures based on di-lysine
(Lys-Lys) peptides, e.g.:
##STR00010##
and tri-lysine peptides (Lys-Lys-Lys), e.g.:
##STR00011##
[0154] In each of the figures above, the indices e, f, f' and f''
represent integers independently selected from 1 to 2500. The
indices q, q' and q'' represent integers independently selected
from 1 to 20.
[0155] In another exemplary embodiment, the conjugates of the
invention include a formula which is a member selected from:
##STR00012##
wherein Q is a member selected from H and substituted or
unsubstituted C.sub.1-C.sub.6 alkyl. The indices e and f are
integers independently selected from 1 to 2500, and the index q is
an integer selected from 0 to 20.
[0156] In another exemplary embodiment, the conjugates of the
invention include a formula which is a member selected from:
##STR00013##
wherein Q is a member selected from H and substituted or
unsubstituted C.sub.1-C.sub.6 alkyl, preferably Me. The indices e,
f and f' are integers independently selected from 1 to 2500, and q
and q' are integers independently selected from 1 to 20.
[0157] In another exemplary embodiment, the conjugate of the
invention includes a structure according to the following
formula:
##STR00014##
wherein the indices m and n are integers independently selected
from 0 to 5000. The indices j and k are integers independently
selected from 0 to 20. A.sup.1, A.sup.2, A.sup.3, A.sup.4, A.sup.5,
A.sup.6, A.sup.7, A.sup.8, A.sup.9, A.sup.10 and A.sup.11 are
members independently selected from H, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted aryl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted heteroaryl, --NA.sup.12A.sup.13, --OA.sup.12 and
--SiA.sup.12A.sup.13. A.sup.12 and A.sup.13 are members
independently selected from substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, and
substituted or unsubstituted heteroaryl.
[0158] In one embodiment according to the formula above, the
branched polymer has a structure according to the following
formula:
##STR00015##
In an exemplary embodiment, A.sup.1 and A.sup.2 are members
independently selected from OCH.sub.3 and OH.
[0159] In another exemplary embodiment, the linker L.sup.a is a
member selected from aminoglycine derivatives. Exemplary polymeric
modifying groups according to this embodiment have a structure
according to the following formulae:
##STR00016##
[0160] In one example, A.sup.1 and A.sup.2 are members
independently selected from OCH.sub.3 and OH. Exemplary polymeric
modifying groups according to this example include:
##STR00017##
[0161] In each of the above embodiment, wherein the modifying group
includes a stereocenter, for example those including an amino acid
linker or a glycerol-based linker, the stereocenter can be either
racemic or defined. In one embodiment, in which such stereocenter
is defined, it has (S) configuration. In another embodiment, the
stereocenter has (R) configuration.
[0162] Those of skill in the art will appreciate that one or more
of the m-PEG arms of the branched polymer can be replaced by a PEG
moiety with a different terminus, e.g., OH, COOH, NH.sub.2,
C.sub.2-C.sub.10-alkyl, etc. Moreover, the structures above are
readily modified by inserting alkyl linkers (or removing carbon
atoms) between the .alpha.-carbon atom and the functional group of
the side chain. Thus, "homo" derivatives and higher homologues, as
well as lower homologues are within the scope of cores for branched
PEGs of use in the present invention.
[0163] The linear and branched PEG conjugates set forth herein may
be prepared using art-recognized methods. Several reviews and
monographs on the functionalization and conjugation of PEG are
available. See, for example, Harris, Macronol. Chem. Phys. C25:
325-373 (1985); Scouten, Methods in Enzymology 135: 30-65 (1987);
Wong et al., Enzyme Microb. Technol. 14: 866-874 (1992); Delgado et
al., Critical Reviews in Therapeutic Drug Carrier Systems 9:
249-304 (1992); Zalipsky, Bioconjugate Chem. 6: 150-165 (1995); and
Bhadra, et al., Pharmazie, 57:5-29 (2002). Routes for preparing
reactive PEG molecules and forming conjugates using the reactive
molecules are known in the art. For example, U.S. Pat. No.
5,672,662, U.S. Pat. No. 6,376,604, WO 99/45964, WO 96/21469, U.S.
Pat. No. 5,932,462, U.S. Pat. No. 5,446,090, WO 99/34833, WO
99/14259, U.S. Pat. No. 6,348,558.
[0164] The art-recognized methods of polymer activation set forth
above are of use in the context of the present invention in the
formation of the branched polymers set forth herein.
Glycosyl Linking Group
[0165] In one embodiment, the modifying group is covalently linked
to the polypeptide via a glycosyl linking group. The saccharide
component of the modified sugar, when interposed between the
polypeptide and a modifying group, becomes a "glycosyl linking
group." In an exemplary embodiment, the glycosyl linking group is
formed from a mono- or oligosaccharide that, after modification
with a modifying group, is a substrate for an appropriate enzyme,
such as a glycosyltransferase. In another exemplary embodiment, the
glycosyl linking group is formed from a glycosyl-mimetic moiety.
The polypeptide conjugates of the invention can include glycosyl
linking groups that are mono- or multi-valent (e.g., antennary
structures). Thus, conjugates of the invention include both species
in which a modifying group is attached to a polypeptide via a
monovalent glycosyl linking group. Also included within the
invention are conjugates in which more than one modifying group is
attached to a polypeptide via a multivalent linking group.
Exemplary linking groups are disclosed in PCT/U.S.07/74139 filed
Jul. 23, 2007, the disclosure of which is incorporated by reference
herein in its entirety.
[0166] In an exemplary embodiment, the invention provides a method
for the isolation of a glycopeptide that is conjugated to a
polymeric modifying moiety through an intact glycosyl linking group
having a formula that is selected from:
##STR00018##
In Formulae I, R.sup.2 is H, CH.sub.2OR.sup.7, COOR.sup.7 or
OR.sup.7, in which R.sup.7 represents H, substituted or
unsubstituted alkyl or substituted or unsubstituted heteroalkyl.
When COOR.sup.7 is a carboxylic acid or carboxylate, both forms are
represented by the designation of the single structure COO.sup.- or
COOH. In Formulae I and II, the symbols R.sup.3, R.sup.4, R.sup.5,
R.sup.6 and R.sup.6' independently represent H, substituted or
unsubstituted alkyl, OR.sup.8, NHC(O)R.sup.9. The index d is 0 or
1. R.sup.8 and R.sup.9 are independently selected from H,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, sialic acid or polysialic acid. At least one of
R.sup.3, R.sup.4, R.sup.5, R.sup.6 or R.sup.6' includes the
polymeric modifying moiety e.g., PEG, linked through a bond or a
linking group. In an exemplary embodiment, R.sup.6 and R.sup.6',
together with the carbon to which they are attached are components
of the pyruvyl side chain of sialic acid. In a further exemplary
embodiment, this side chain is functionalized with the polymeric
modifying moiety. In another exemplary embodiment, R.sup.6 and
R.sup.6', together with the carbon to which they are attached are
components of the side chain of sialic acid and the polymeric
modifying moiety is a component of R.sup.5.
[0167] In a further exemplary embodiment, the polymeric modifying
moiety is bound to the sugar core, generally through a heteroatom,
e.g, nitrogen, on the core through a linker, L, as shown below:
##STR00019##
R.sup.1 is the polymeric moiety and L is selected from a bond and a
linking group. The index w represents an integer selected from 1-6,
preferably 1-3 and more preferably 1-2. Exemplary linking groups
include substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl moieties and sialic acid. An exemplary
component of the linker is an acyl moiety.
[0168] An exemplary compound according to the invention has a
structure according to Formulae I or II, in which at least one of
R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6 or R.sup.6' has the
formula:
##STR00020##
[0169] In another example according to this embodiment at least one
of R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6 or R.sup.6' has the
formula:
##STR00021##
in which s is an integer from 0 to 20 and R.sup.1 is a linear
polymeric modifying moiety.
[0170] In an exemplary embodiment, the polymeric modifying
moiety-linker construct is a branched structure that includes two
or more polymeric chains attached to central moiety. In this
embodiment, the construct has the formula:
##STR00022##
in which R.sup.1 and L are as discussed above and w' is an integer
from 2 to 6, preferably from 2 to 4 and more preferably from 2 to
3.
[0171] When L is a bond it is formed between a reactive functional
group on a precursor of R.sup.1 and a reactive functional group of
complementary reactivity on the saccharyl core. When L is a
non-zero order linker, a precursor of L can be in place on the
glycosyl moiety prior to reaction with the R.sup.1 precursor.
Alternatively, the precursors of R.sup.1 and L can be incorporated
into a preformed cassette that is subsequently attached to the
glycosyl moiety. As set forth herein, the selection and preparation
of precursors with appropriate reactive functional groups is within
the ability of those skilled in the art. Moreover, coupling the
precursors proceeds by chemistry that is well understood in the
art.
[0172] In an exemplary embodiment, L is a linking group that is
formed from an amino acid, or small peptide (e.g., 1-4 amino acid
residues) providing a modified sugar in which the polymeric
modifying moiety is attached through a substituted alkyl linker.
Exemplary linkers include glycine, lysine, serine and cysteine. The
PEG moiety can be attached to the amine moiety of the linker
through an amide or urethane bond. The PEG is linked to the sulfur
or oxygen atoms of cysteine and serine through thioether or ether
bonds, respectively.
[0173] In an exemplary embodiment, R.sup.5 includes the polymeric
modifying moiety. In another exemplary embodiment, R.sup.5 includes
both the polymeric modifying moiety and a linker, L, joining the
modifying moiety to the remainder of the molecule. As discussed
above, L can be a linear or branched structure. Similarly, the
polymeric modifying can be branched or linear.
[0174] In one embodiment, the present invention provides methods
for the isolation of an erythropoietin peptide conjugate comprising
the moiety:
##STR00023##
wherein D is a member selected from --OH and R.sup.1-L-HN--; G is a
member selected from H and R.sup.1-L- and
--C(O)(C.sub.1-C.sub.6)alkyl; R.sup.1 is a moiety comprising a
straight-chain or branched poly(ethylene glycol) residue; and L is
a linker, e.g., a bond ("zero order"), substituted or unsubstituted
alkyl and substituted or unsubstituted heteroalkyl. In exemplary
embodiments, when D is OH, G is R.sup.1-L-, and when G is
C(O)(C.sub.1-C.sub.6)alkyl, D is R.sup.1-L-NH--.
[0175] In another exemplary embodiment, the invention provides a
conjugate formed between a modified sugar of the invention and a
substrate EPO peptide. In this embodiment, the sugar moiety of the
modified sugar becomes a glycosyl linking group interposed between
the peptide substrate and the modifying group. An exemplary
glycosyl linking group is an intact glycosyl linking group, in
which the glycosyl moiety or moieties forming the linking group are
not degraded by chemical (e.g., sodium metaperiodate) or enzymatic
(e.g., oxidase) processes. Selected conjugates of the invention
include a modifying group that is attached to the amine moiety of
an amino-saccharide, e.g., mannosamine, glucosamine, galactosamine,
sialic acid etc. Exemplary modifying group-intact glycosyl linking
group cassettes according to this motif are based on a sialic acid
structure, such as those having the formulae:
##STR00024##
[0176] In the formulae above, R.sup.1 and L are as described above.
Further detail about the structure of exemplary R.sup.1 groups is
provided below.
[0177] In still a further exemplary embodiment, the conjugate is
formed between a substrate EPO and a saccharyl moiety in which the
modifying group is attached through a linker at the 6-carbon
position of the saccharyl moiety. Thus, illustrative conjugates
according to this embodiment have the formula:
##STR00025##
in which the radicals are as discussed above. Such saccharyl
moieties include, without limitation, glucose, glucosamine,
N-acetyl-glucosamine, galactose, galactosamine,
N-acetyl-galactosamine, mannose, mannosamine, N-acetyl-mannosamine,
and the like.
[0178] Due to the versatility of the methods available for
modifying glycosyl residues on a therapeutic peptide such as EPO,
the glycosyl structures on the peptide conjugates of the invention
can have substantially any structure. Moreover, the glycans can be
O-linked or N-linked. As exemplified in the discussion below, each
of the pyranose and furanose derivatives discussed above can be a
component of a glycosyl moiety of a peptide.
[0179] The invention provides a modified EPO peptide that includes
a glycosyl group having the formula:
##STR00026##
[0180] In other embodiments, the group has the formula:
##STR00027##
in which the index t is 0 or 1.
[0181] In a still further exemplary embodiment, the group has the
formula:
##STR00028##
in which the index t is 0 or 1.
[0182] In yet another embodiment, the group has the formula:
##STR00029##
in which the index p represents and integer from 1 to 10; and a is
either 0 or 1.
[0183] In an exemplary embodiment, a glycoPEGylated EPO peptide of
the invention includes at least one N-linked glycosyl residue
selected from the glycosyl residues set forth below:
##STR00030##
[0184] In the formulae above, the index t is 0 or 1 and the index p
is an integer from 1 to 10. The symbol R.sup.15' represents H, OH
(e.g., Gal-OH), a sialyl moiety, a polymer modified sialyl moiety
(i.e., glycosyl linking group-polymeric modifying moiety
(Sia-L-R.sup.1)) or a sialyl moiety to which is bound a polymer
modified sialyl moiety (e.g., Sia-Sia-L-R.sup.1) ("Sia-Sia.sup.p").
An exemplary EPO peptide of the invention will include at least one
glycan having a R.sup.15'. The oxygen, with the open valence, of
Formulae I and II is preferably attached through a glycosidic
linkage to a carbon of a Gal or GalNAc moiety. In a further
exemplary embodiment, the oxygen is attached to the carbon at
position 3 of a galactose residue. In an exemplary embodiment, the
modified sialic acid is linked .alpha.2,3- to the galactose
residue. In another exemplary embodiment, the sialic acid is linked
.alpha.2,6- to the galactose residue.
[0185] The modified glycan is bound to one or more position
selected from Asn 24, Asn 38, Asn 83 and/or Ser 126. In an
exemplary embodiment, the EPO is derived from mammalian cells and
the modifying group is only on the glycan at Asn 24. In one
embodiment according to this motif, the glycosyl linking moiety is
linked to a Sia residue through another Sia residue, e.g.:
##STR00031##
An exemplary species according to this motif is prepared by
conjugating Sia-L-R.sup.1 to a terminal sialic acid of the glycan
at Asn 24 using an enzyme that forms Sia-Sia bonds, e.g., CST-II,
ST8Sia-II, ST8Sia-III and ST8Sia-IV.
[0186] In another exemplary embodiment, the glycans have a formula
that is selected from the group:
##STR00032##
and a combination thereof.
[0187] The glycans of this group generally correspond to those
found on an EPO peptide that is produced by insect cells (e.g.,
Sf9), followed by remodeling of the glycan and glycoPEGylation
according to the methods set forth herein. For example,
insect-derived EPO that is expressed with a tri-mannosyl core is
subsequently contacted with a GlcNAc donor and a GlcNAc transferase
and a Gal donor and a Gal transferase. Appending GlcNAc and Gal to
the tri-mannosyl core is accomplished in either two steps or a
single step. A modified sialic acid is added to at least one branch
of the glycosyl moiety as discussed herein. Those Gal moieties that
are not functionalized with the modified sialic acid are optionally
"capped" by reaction with a sialic acid donor in the presence of a
sialyl transferase.
[0188] In an exemplary embodiment, at least 60% of terminal Gal
moieties in a population of peptides is capped with sialic acid,
preferably at least 70%, more preferably, at least 80%, still more
preferably at least 90% and even more preferably at least 95%, 96%,
97%, 98% or 99% are capped with sialic acid.
[0189] In each of the formulae above, R.sup.15' is as discussed
above. Moreover, an exemplary modified EPO peptide of the invention
will include at least one glycan with an R.sup.15' moiety having a
structure according to Formulae I or II.
[0190] In another exemplary embodiment, the EPO is derived from
insect cells, which are remodeled by adding GlcNAc and Gal to the
mannose core. The remodeled peptide is glycopegylated using a
sialic acid bearing a linear PEG moiety, affording an EPO peptide
that comprises at least one moiety having the formula:
##STR00033##
in which s represents and integer from 1 to 10; and f represents
and integer from 1 to 2500.
[0191] In certain embodiments, the EPO peptide includes three such
moieties, one attached at each of Asn 24, Asn 38 and Asn 83. In
another embodiment, the peptide includes two such moieties attached
at a combination of two of these Asn moieties. There is also
provided a composition that is a mixture of these two species
(i.e., PEG.sub.3 and PEG.sub.2). The mixture preferably includes at
least 75%, preferably at least 80%, more preferably at least 85%,
still more preferably 90% and even more preferably 95%, 96%, 97% or
98% of the species that includes the three modified glycosyl
residues. Unmodified terminal Gal residues are optionally capped
with Sia as discussed above. In an exemplary embodiment, the
peptide is expressed in insect cells, remodeled and
glycopegylated.
[0192] The indices e and q are as discussed above. In an exemplary
embodiment, e for each of the modified glycosyl moieties is an
integer that provides as PEG moiety having a molecular weight of
approximately 10 kDa.
I.b) Isolation of Polypeptide Conjugates
[0193] The membrane filtration and chromatographic techniques
described below are useful steps within the methods of the
invention and apply to the isolation of polypeptide conjugates, in
which a polypeptide is linked to at least one modifying group, such
as a poly(alkylene oxide) moiety. It is to be understood that
unless the order of steps is explicitly recited, the exemplary
steps can be performed in any desired order.
Membrane Filtration
[0194] In one embodiment, the methods of the invention includes one
or more membrane filtration steps. Membrane filtration is a
separation technique widely used for clarifying, concentrating, and
purifying polypeptides.
Ultrafiltration/Diafiltration
[0195] Ultrafiltration Using a Membrane with a Small MWCO
[0196] In one exemplary embodiment, the polypeptide purification
process of the present invention includes at least one
diafiltration/ultrafiltration step, e.g., as the final process step
to generate a composition suitable for storage. In one example,
diafiltration/ultrafiltration is performed to condition a mixture
for a chromatographic process step. For example, the eluate from a
hydrophobic interaction chromatography step is concentrated and the
buffer is exchanged to prepare the sample for the next purification
step (e.g., cation exchange chromatography).
[0197] In an exemplary embodiment, the diafiltration step is
employed to concentrate the sample. In another exemplary embodiment
the diafiltration step is employed to alter the buffer. In yet
another exemplary embodiment, the new buffer is suitable for
storage of the purified polypeptide conjugate. The
diafiltration/ultrafiltration membrane can have any molecular
weight cuttoff (MWCO) specification.
[0198] In an exemplary embodiment, the feed is passed through an
ultrafiltration membrane with a MWCO suitable to concentrate the
purified polypeptide conjugate. The selected MWCO will depend on
the combined size of the polypeptide and the modifying group, such
as the size of a poly(alkylene oxide) moiety covalently linked to
the polypeptide. To concentrate a sample, the membrane is chosen to
have a MWCO that is substantially lower than the molecular weight
of the purified peptide conjugate. In one example, the
ultrafiltration membrane is selected to have a MWCO that is 3 to 6
times lower than the molecular weight of the peptide conjugate to
be retained by the membrane. If the flow rate or the processing
time is of major consideration, selection of a membrane with a MWCO
toward the lower end of this range (e.g. 3.times.) will yield
higher flow rates. If recovery of peptide conjugate is the primary
concern, a tighter membrane (e.g. 6.times.) is selected (typically
with a slower flow rate).
[0199] In one exemplary embodiment, the diafiltration membrane has
a MWCO of about 2 kDa to about 500 kDa. In another exemplary
embodiment, the diafiltration membrane has a MWCO of about 5 kDa to
about 400 kDa, about 5 kDa to about 300 kDa or about 5 kDa to about
200 kDa. In yet another exemplary embodiment, the diafiltration
membrane has a MWCO of about 5 kDa to about 180 kDa, 5 kDa to about
160 kDa, 5 kDa to about 140 kDa, 5 kDa to about 130 kDa, 5 kDa to
about 120 kDa, 5 kDa to about 110 kDa, or 5 kDa to 100 kDa. When
the polypeptide conjugate is EPO-PEG, the diafiltration membrane
has a MWCO of about 5 kDa to about 80 kDa, 5 kDa to about 60 kDa, 5
kDa to about 40 kDa or 5 kDa to about 20 kDa. In one embodiment,
when the polypeptide conjugate is EPO-[PEG-10 kDa].sub.3, the
diafiltration membrane has a MWCO of about 8 kDa to about 12 kDa
and preferably about 10 kDa.
[0200] In another exemplary embodiment, filtration is effected
using a transmembrane pressure between about 1 and about 30 psi and
a filter membrane with a MWCO of between about 5 kDa to about 15
kDa, and preferably 10 kDa. The filtration step produces a
retentate stream and a permeate stream. The retentate may be
recycled to a reservoir for the peptide solution feed under
conditions of essentially constant peptide concentration in the
feed by adding a buffer solution to the retentate.
[0201] The surface area of the filtration membrane used will
generally depend on the amount of peptide conjugate to be purified.
The membrane may be made of a low-binding material to minimize
adsorptive losses and is preferably durable, cleanable, and
chemically compatible with the buffers to be used. A number of
suitable membranes are commercially available. In an exemplary
embodiment, the ultrafiltration/diafiltration membrane is a member
selected from cellulose acetate, regenerated cellulose and
polyethersulfone. Suitable membranes include those, in which the
membrane polymer is chemically modified. In a preferred embodiment,
the membrane is regenerated cellulose.
[0202] In one embodiment, the flow rate is adjusted to maintain a
constant transmembrane pressure. Generally, filtration will proceed
faster with higher pressures and higher flow rates, but higher flow
rates may also result in damage to the peptide or loss of peptide
due to passage through the membrane. In addition, various devices
may have certain pressure limitations on their operation, and the
pressure is adjusted according to the manufacturer's specification.
In an exemplary embodiment, the pressure is between about 1 to
about 30 psi, and in another exemplary embodiment the pressure is
between about 8 psi to about 15 psi. Typically, the circulation
pump is a peristaltic pump or diaphragm pump in the feed channel
and the pressure is controlled by adjusting the retentate
valve.
[0203] Subsequent to a filtration step, the retentate is collected.
Water or an aqueous buffer (e.g. diafiltration buffer) may be used
to wash the membrane filter and recover any peptide retained by the
membrane. The wash liquid may be combined with the original
retentate containing the concentrated peptide. The retentate is
optionally dialyzed against another buffer, such as TRIS or
HEPES.
[0204] The purified product is stored at a low temperature. In an
exemplary embodiment the product is stored at about -20.degree. C.
at a polypeptide concentration of about 1 mg to about 10 mg of
peptide conjugate per mL storage buffer. Before storage the product
solution maybe sterile filtered, e.g., using a membrane filter
having a pore size of about 2 .mu.M (e.g., cellulose acetate
filter).
Chromatographic Isolation of Polypeptide Conjugates
[0205] A variety of recognized chromatographic techniques, such as
size exclusion chromatography (gel filtration), ion exchange
chromatography, hydrophobic interaction chromatography (HIC),
affinity chromatography, mixed-mode chromatography, hydroxyapatite
and fluoroapatite chromatography are used for the isolation of
polypeptides and proteins. These technologies can also be used to
isolate polypeptide conjugates. In an exemplary embodiment, methods
of the invention employ a combination of several chromatographic
techniques. The order in which these steps are performed is
dependent on the nature of the polypeptide conjugate being purified
and the nature of the contaminants to be removed.
[0206] Suitable techniques for the practice of the invention
separate the polypeptide conjugate of interest from a variety of
contaminants on the basis of charge, degree of hydrophobicity,
and/or size. Different chromatographic resins and membranes are
available for each of these techniques, allowing accurate tailoring
of the purification scheme.
[0207] In one chromatographic technique, the components in a
mixture interact differently with the column material and move at
different rates along the column length, achieving a physical
separation that increases as the components pass through the
column. In another chromatographic technique, components of the
mixture, including the peptide conjugate of interest, adhere
selectively to the separation medium, while other components are
found in the flow-through. The initially retained components are
then eluted differentially by varying the composition of the
solvent or buffer system. In another approach, the desired
components are found in the flow-through while impurities are
retained on the column and thus removed from the mixture.
Ion Exchange Chromatography
[0208] In one embodiment, the methods of the invention employ at
least one ion exchange chromatography step. Anion and cation
exchange chromatography are known in the art. Ion exchange
chromatography separates compounds based on their net charge. Ionic
molecules are classified as either anions (having a negative
charge) or cations (having a positive charge). Some molecules
(e.g., proteins) may have both anionic and cationic groups. A
positively charged support (anion exchanger) will bind a compound
with an overall negative charge. Conversely, a negatively charged
support (cation exchanger) will bind a compound with an overall
positive charge. Ion exchange matrices can be further categorized
as either strong or weak exchangers. Strong ion exchange matrices
are charged (ionized) across a wide range of pH levels. Weak ion
exchange matrices are ionized within a narrow pH range. The ionic
groups of exchange columns are covalently bound to the gel matrix
and are compensated by small concentrations of counter ions, which
are present in the buffer. The most common ion exchange chemistries
include: quaternary ammonium residues (Q) for strong anion
exchange, diethylaminoethyl residues (DEAE) for weak anion
exchange, sulfopropyl (SP) resins and sulfonic acid (S) resins for
strong cation exchange and carboxymethyl residues (CM) for weak
cation exchange.
[0209] When adding a sample to the column, an exchange with the
weakly bound counter ions takes place. The size of the sample
volume in ion exchange chromatography is of secondary importance as
long as the initial solvent is of low eluting strength, to not
allow the sample components to proceed through the column. Under
such conditions, the sample components are preferably collected at
the top of the column. When the gradient is begun with the addition
of a stronger eluting mobile phase, the sample components begin
their separation. If poor separation is observed, it might be
improved by a change in the gradient slope. If the polypeptide
conjugate does not bind to the column under the selected
conditions, the composition and/or the pH of the starting buffer
should be changed. The buffer system can further be optimized by
choosing different buffer salts since each buffer composition
solvates the ion exchanger and the sample components uniquely.
[0210] In one example, any conventional buffer system with a salt
concentration of about 5 mM up to about 50 mM can be used for ion
exchange chromatography. However, positively charged buffering ions
are used for anion exchangers and negatively charged ones are used
for cation exchangers. Phosphate buffers are generally used on both
exchanger types. Typically, the highest salt concentration that
permits binding of the peptide of interest is used as the starting
condition. In one example, all buffers are prepared from
MilliQ-water and filtered (0.45 or 0.22 .mu.m filter).
Anion Exchange Chromatography
[0211] In an exemplary embodiment a sample containing the peptide
conjugate of interest is loaded onto an anion exchanger in a
loading buffer comprising a salt concentration below the
concentration at which the peptide would elute from the column. The
pH of the buffer is selected so that the purified peptide is
retained on the anion exchange medium. Changing the pH of the
buffer alters the charge of the peptide, and lowering the pH value
shortens the retention time with anion exchangers. The isoelectric
point (pI) of a protein is the pH at which the charge of a protein
is zero. Typically, with anion exchangers the pH value of the
buffer is kept 1.5 to 2 times higher than the pI value of the
peptide of interest. Alternatively, the anion exchange conditions
are selected to preferentially bind impurities, while the purified
peptide is found in the flow-through.
[0212] The column may be washed with several column volumes (CV) of
buffer to remove unbound substances and/or those substances that
bind weakly to the resin. Fractions are then eluted from the column
using, for example, a saline gradient according to conventional
methods. The salt in the solution competes with the protein in
binding to the column and the protein is released. Components with
weak ionic interactions elute at a lower salt concentration than
components with a strong ionic interaction. Sample fractions are
collected from the column. Fractions containing high levels of the
desired peptide and low levels of impurities are pooled or
processed separately.
[0213] In one example, anion exchange used in the process of the
current invention is employed to isolate the polypeptide conjugate
from contaminants such as particulates, chemicals and
proteins/peptides (e.g., enzymes used in a glycoPEGylation
reaction).
[0214] Anion exchange media are known to those of skill in the art.
Exemplary anion exchange media are described, e.g., in Protein
Purification Methods, A Practical Approach, Ed. Harris E L V, Angal
S, IRL Press Oxford, England (1989); Protein Purification, Ed.
Janson J C, Ryden L, VCH-Verlag, Weinheim, Germany (1989); Process
Scale Bioseparations for the Biopharmaceutical Industry, Ed. Shukla
A A, Etzel M R, Gadam S, CRC Press Taylor & Francis Group
(2007), pages 188-196; Protein Purification Handbook, GE Healthcare
2007 (18-1132-29) and Protein Purification, Principles, High
Resolution Methods and Applications (2.sup.nd Edition 1998), Ed.
Janson J-C and Ryden L, the disclosures of which are incorporated
herein by reference in their entirety. An exemplary anion exchanger
of the invention is selected from quaternary ammonium filters and
DEAE resins. In one embodiment, the anion exchanger is a quaternary
ammonium resin (e.g. Mustang Q ion exchange membrane, Pall
Corporation). In one example, the anion exchanger is Sartobind Q.
Other useful resins include QXL, Capto and BigBeads resins.
[0215] Exemplary anion exchange media are summarized below:
GE Healthcare:
Q-Sepharose FF
Q-Sepharose BB
Q-Sepharose XL
Q-Sepharose HP
Mini Q
Mono Q
Mono P
DEAE Sepharose FF
Source 15Q
Source 30Q
Capto Q
[0216] ANX Sepharose 4 FF (high sub)
Streamline DEAE
Streamline QXL
Applied Biosystems:
[0217] Poros HQ 10 and 20 um self pack Poros HQ 20 and 50 um bulk
media
Poros PI 20 and 50 um
Poros D 50 um
Tosohaas:
Toyopearl DEAE 650S, M and C
Super Q 650
QAE 550C
Pall Corporation:
DEAE Hyper D
Q Ceramic Hyper D
[0218] Mustang Q membrane absorber
Merck KGgA:
Fractogel DMAE
FractoPrep DEAE
Fractoprep TMAE
Fractogel EMD DEAE
Fractogel EMD TMAE
Sartorious:
[0219] Sartobind Q membrane absorber
Cation Exchange Chromatography
[0220] In one embodiment, the method of the invention includes at
least one cation exchange step. In an exemplary embodiment a sample
containing the peptide conjugate of interest is loaded onto a
cation exchange resin in a loading buffer comprising a salt
concentration below the concentration at which the peptide would
elute from the column.
[0221] In one example, the pH of the loading buffer is selected so
that the peptide conjugate of interest is retained on the cation
exchange resin. Changing the pH of the buffer alters the charge of
the peptide and increasing the pH of the buffer shortens the
retention times with cation exchangers. Typically, cation exchanges
are performed at 1.5 to 2 pH units below the peptide's pI.
Alternatively, the cation exchange conditions are selected to
preferentially bind impurities, while the purified peptide is found
in the flow-through.
[0222] In one embodiment, the column is washed with several column
volumes of buffer to remove unbound substances and those substances
that bind weakly to the resin. Fractions are then eluted from the
column using a salt gradient according to conventional methods.
Sample fractions are collected from the column. One or more
fraction containing high levels of the desired peptide and low
levels of impurities are collected, and optionally pooled.
[0223] In an exemplary embodiment the cation exchangers used in the
process of the current invention provide one of the primary
purification steps of the purification process. In one embodiment,
the cation exchanger removes undesired proteins from the mixture,
which contains the peptide conjugate of interest. In another
embodiment, the cation exchange step is useful to remove unwanted
glycoforms of the purified polypeptide conjugate.
[0224] Cation exchange media are known to those of skill in the
art. Exemplary cation exchange media are described, e.g., in
Protein Purification Methods, A Practical Approach, Ed. Harris E L
V, Angal S, IRL Press Oxford, England (1989); Protein Purification,
Ed. Janson J C, Ryden L, VCH-Verlag, Weinheim, Germany (1989);
Process Scale Bioseparations for the Biopharmaceutical Industry,
Ed. Shukla A A, Etzel M R, Gadam S, CRC Press Taylor & Francis
Group (2007), pages 188-196; Protein Purification Handbook, GE
Healthcare 2007 (18-1132-29) and Protein Purification, Principles,
High Resolution Methods and Applications (2.sup.nd Edition 1998),
Ed. Janson J-C and Ryden L, the disclosures of which are are
incorporated herein by reference in their entirety. In an exemplary
embodiment, cation exchange resins of use in the invention are
selected from sulfonic acid (S) and carboxymethyl (CM) supports. In
one embodiment, the cation exchanger is a sulfonic acid support
(e.g. UNOsphereS, Bio-Rad Laboratories) or a sulphopropyl (SP)
resin. In another embodiment, the cation exchange resin is selected
from SPFF, SPHP sepharose, BigBeads SP, Capto S and the like. In
one example, the cation exchanger is Source 15S.
[0225] Exemplary commercial cation exchange media are summarized
below:
GE Healthcare:
SP-Sepharose FF
SP-Sepharose BB
SP-Sepharose XL
SP-Sepharose HP
Mini S
Mono S
CM Sepharose FF
Source 15S
Source 30S
Capto S
MacroCap SP
Streamline SP-XL
Streamline CST-1
Tosohaas Resins:
Toyopearl Mega Cap II SP-550 EC
Toyopearl Giga Cap S--650M
Toyopearl 650S, M and C
Toyopeal SP650S, M, and C
Toyopeal SP550C
JT Baker Resins:
Carboxy-Sulphon--5, 15 and 40 um
Sulfonic--5, 15, and 40 um
Applied Biosystems:
Poros HS 20 and 50 um
Poros S10 and 20 um
Pall Corp:
S Ceramic Hyper D
CM Ceramic Hyper D
Merck KGgA Resins:
Fractogel EMD SO3
Fractogel EMD COO--
Fractogel EMD SE Hicap
Fracto Prep So3
Biorad Resin:
Unosphere S
Sartorius Membrane:
[0226] Sartobind S membrane absorber
[0227] The ion exchangers used in the methods of the invention are
optionally membrane adsorbers rather than chromatographic resins or
supports. In an exemplary embodiment, the membrane adsorber is a
cation exchanger. In another exemplary embodiment the membrane
adsorber is a sulfonic acid (S) cation exchanger (e.g. Sartobind S,
Sartorius AG). The membrane adsorber is optionally disposable.
Mixed-Mode or Pseudo-Affinity Chromatography
[0228] In one embodiment, the peptide conjugate purification
process of the invention includes mixed-mode or pseudo-affinity
chromatography.
[0229] In one example, the process involves chromatography
performed on ceramic or crystalline apatite media, such as
hydroxyapatite (HA) chromatography and fluoroapatite (FA)
chromatography. HA and FA chromatography are effective purification
mechanisms, providing biomolecule selectivity, complementary to ion
exchange and/or hydrophobic interaction techniques. Hydroxyapatite
and fluoroapatite chromatography are known in the art. In one
example, the apatite medium is Adhere MMC.
Hydroxyapatite
[0230] Exemplary hydroxyapatite sorbents of type I and type II are
selected from ceramic and crystalline materials. Hydroxyapatite
sorbents are available in different particle sizes (e.g. type 1,
Bio-Rad Laboratories). In an exemplary embodiment, the particle
size of the hydroxyapatite sorbent is between about 20 .mu.m and
about 180 .mu.m, between about 20 .mu.m and about 100 .mu.m or
between about 60 .mu.m and about 100 .mu.m. In a particular
example, the particle size of the hydroxyapatite sorbent is about
80 .mu.m.
[0231] In one embodiment, the hydroxyapatite sorbent is composed of
cross-linked agarose beads with microcrystals of hydroxyapatite
entrapped in the agarose mesh. Optionally, the agarose is
chemically stabilized (e.g. with epichlorohydrin under strongly
alkaline conditions). In one exemplary embodiment, the
hydroxyapatite sorbent is HA Ultrogel (Pall Corporation).
Fluoroapatite
[0232] Exemplary type I and type II fluoroapatite sorbents are
selected from ceramic (e.g., bead-like particles) and crystalline
materials. Ceramic fluoroapatite sorbents are available in
different particle sizes (e.g. type 1 and type 2, Bio-Rad
Laboratories). In an exemplary embodiment the particle size of the
ceramic fluoroapatite sorbent is from about 20 .mu.m to about 180
.mu.m, preferably about 20 to about 100 .mu.m, more preferably
about 20 .mu.m to about 80 .mu.m. In one example, the particle size
of the ceramic fluoroapatite medium is about 40 .mu.m (e.g., type 1
ceramic fluoroapatite). In another example, the fluoroapatite
medium includes hydroxyapatite in addition to fluoroapatite. In a
particular example, the fluoroapatite medium is Bio-Rad's CFT.TM.
Ceramic Fluoroapatite.
[0233] The selection of the flow velocity used for loading the
sample onto the hydroxyapatite or fluoroapatite column, as well as
the elution flow velocity depends on the type of hydroxyapatite or
fluoroapatite sorbent and on the column geometry. In one exemplary
embodiment, at process scale, the loading flow velocity is selected
from about 30 to about 900 cm/h, from about 150 to about 900 cm/h,
preferably from about 500 to about 900 cm/h and, more preferably,
from about 600 to about 900 cm/h.
[0234] In an exemplary embodiment, the pH of the elution buffer is
selected from about pH 7 to about pH 9, and preferably from about
pH 7.5 to about pH 8.0.
[0235] In one aspect the present invention provides a method of
purifying a recombinant peptide by hydroxyapatite or fluoroapatite
chromatography. The method includes the following steps: (a)
desalting a mixture containing the peptide, forming a desalted
mixture (e.g. by gel filtration) that has a salt conductivity,
which is sufficiently low to increase the peptide-binding capacity
of the hydroxyapatite or fluoroapatite resin; (b) applying the
desalted mixture to a hydroxyapatite or fluoroapatite resin; (c)
washing the hydroxyapatite or fluoroapatite resin, thereby eluting
unwanted components from the resin; (d) eluting the peptide from
the resin with an elution buffer that optionally contains an amino
acid; and (e) collecting one or more eluate fraction containing the
peptide.
Desalting
[0236] In one embodiment, the mixture containing the peptide of
interest is desalted prior to subjecting the mixture to HA or
fluoroapatite chromatography. The desalting step increases the
capacity of the apatite column to bind the peptide of interest. In
one embodiment, the apatite column capacity (amount of peptide per
liter of resin), increases with decreasing salt conductivity of the
load, which contains the peptide.
[0237] In an exemplary embodiment, in which the load is desalted,
the mass loading of peptide per liter of HA resin is from about 1
to about 25 g/L, from about 1 to about 20 g/L, preferably from
about 1 to about 15 g/L and more preferably from about 1 to about
10 g/L.
[0238] In another exemplary embodiment, the conductivity of the
load can be decreased using a method selected from desalting and
diluting.
[0239] In an exemplary embodiment, the conductivity of the loading
buffer is lowered by desalting and preferred conductivities are
from about 0.1 to about 4.0 mS/cm, preferably from about 0.1 to
about 1.0 mS/cm, more preferably from about 0.1 to about 0.6 mS/cm
and, still more preferably, from about 0.1 to about 0.4 mS/cm.
[0240] In one example, desalting of peptide conjugate solutions is
achieved using membrane filters wherein the membrane filter has a
MWCO smaller than the peptide/protein of interest. The
peptide/protein is found in the retentate and is reconstituted in a
buffer of choice. However, when purifying peptides of relatively
low molecular weight (e.g. EPO), the MWCO of the membrane used for
desalting must be relatively small in order to avoid leaking of the
peptide through the membrane pores. However, filtering a large
volume of liquid through a small MWCO membrane (e.g. with a pore
size of about 5 kDa), typically requires large membrane areas and
the filtering process is time consuming.
[0241] Therefore, in one embodiment, desalting of the HA or
fluoroapatite chromatography load is accomplished using
size-exclusion chromatography (e.g. gel filtration). The technique
separates molecules on the basis of size. Typically, high molecular
weight components can travel through the column more easily than
smaller molecules, since their size prevents them from entering
bead pores. Accordingly, low-molecular weight components take
longer to pass through the column. Thus, low molecular weight
materials, such as unwanted salts, can be separated from the
peptide of interest.
[0242] In an exemplary embodiment, the column material is selected
from dextran, agarose, and polyacrylamide gels, in which the gels
are characterized by different particle sizes. In another exemplary
embodiment, the material is selected from rigid, aqueous-compatible
size exclusion materials. An exemplary gel filtration resin of the
invention is Sepharose G-25 resin (GE Healthcare).
[0243] In an exemplary embodiment, desalting is performed
subsequent to cation exchange chromatography (e.g. after Source 15S
chromatography).
Addition of an Amino Acid to the Elution Buffer
[0244] In one embodiment, an amino acid is added to the elution
buffer, which is used to elute the peptide of interest from the HA
or fluoroapatite resin. In an exemplary embodiment the amino acid
is added to the elution buffer at a final concentration of about 5
mM to about 50 mM, about 10 mM to about 40 mM, preferably about 15
mM to about 30 mM and, more preferably, about 20 mM.
[0245] In one embodiment, the addition of an amino acid (e.g.
glycine) to the elution buffer increases the step recovery of
peptide from HA chromatography when compared to the recovery
obtained without the addition of an amino acid. In an exemplary
embodiment, the recovery of peptide is increased by addition of the
amino acid at least about 1% to about 20%, by at least about 1% to
about 15%, by at least about 1% to about 10%, preferably by at
least about 1% to about 7% and, more preferably, by about 5%.
[0246] In another exemplary embodiment, the addition of an amino
acid (e.g. glycine) causes the elution peak of the purified peptide
to be sharper. Thus, less peptide is recovered in the tail
fractions of the peak and more peptide is recovered in the main
peak. In another exemplary embodiment, the addition of an amino
acid (e.g. glycine) does not decrease the purity of the product
from HA chromatography.
[0247] In an exemplary embodiment, the amino acid is glycine. In a
preferred embodiment, glycine is added to the elution buffer at a
final concentration of 20 mM.
Hydrophobic Interaction Chromatography (HIC)
[0248] Hydrophobic interaction chromatography (HIC) is a liquid
chromatography technique that separates biomolecules based on
differences in their surface hydrophobicity. For example,
hydrophobic amino acid side chains exposed on the surface of a
polypeptide, can interact with hydrophobic moieties on the HIC
matrix. The amount, of exposed hydrophobic amino acids differs
between polypeptides and so does the ability of polypeptides to
interact with HIC gels. Hydrophobic interaction between a
biomolecule and the HIC matrix is typically enhanced by high ionic
strength buffers, and of biomolecules is most often performed at
high salt concentrations. The elution of the peptide of interest
from the column is then initiated by decreasing salt gradients.
[0249] In one embodiment, the HIC resin is selected for optimal
resolution of different polypeptide glycoforms. Exemplary HIC
resins useful in the methods of the invention are described, e.g.,
in Protein Purification Methods, A Practical Approach, Ed. Harris
ELV, Angal S, IRL Press Oxford, England (1989) page 224, Protein
Purification, Ed. Janson J C, Ryden L, VCH-Verlag, Weinheim,
Germany (1989) pages 207-226, Process Scale Bioseparations for the
Biopharmaceutical Industry, Ed. Shukla A A, Etzel M R, Gadam S, CRC
Press Taylor & Francis Group (2007), pages 197-206, Hydrophobic
Interaction and Reversed Phase Chromatography, Principles and
Methods, GE Healthcare 2007 (11-0012-69), Protein Purification
Handbook, GE Healthcare 2007 (18-1132-29) and Protein Purification,
Principles, High Resolution Methods and Applications (2.sup.nd
Edition 1998), Ed. Janson J-C and Ryden L, "Hydrophobic Interaction
Chromatography, page 283, the disclosures of which are incorporated
herein by reference in their entirety.
[0250] HIC media are distinguished by the hydrophobic moiety that
they carry, by the particle size (e.g. bead size), the pore size
and the density of the hydrophobic moieties on the HIC matrix (e.g.
low substitution or high substitution). In an exemplary embodiment,
the hydrophobic moieties of the column matrix are members selected
from alkyl groups, aromatic groups and ethers. Exemplary
hydrophobic alkyl groups include lower alkyl groups, such as
n-propyl, isopropyl, n-butyl, iso-butyl, and n-octyl. Exemplary
aromatic groups include substituted and unsubstituted phenyl.
[0251] In another exemplary embodiment the matrix of the HIC medium
is a member selected from agarose, sepharose (GE Healthcare),
polystyrene, divinylbenzene, and combinations thereof. Exemplary
HIC resins include Butyl Fast Flow and Phenyl Fast Flow (e.g., GE
Healthcare) in either low or high substituted versions. In a
preferred embodiment, the HIC resin is a phenyl resin. In one
particular example, the HIC resin is Phenyl650S or Phenyl650M
(e.g., Tosohaas, Toyopearl).
[0252] In one example, the HIC medium is selected from the
following commercial resins:
GE Healthcare HIC Resins:
Butyl Sepharose 4 FF
Butyl-S Sepharose FF
Octyl Sepharose 4 FF
Phenyl Sepharose BB
Phenyl Sepharose HP
Phenyl Sepharose 6 FF High Sub
Phenyl Sepharose 6 FF Low Sub
Source 15ETH
Source 151SO
Source 15PHE
[0253] Capto Phenyl (prototype resin) Capto Butyl (prototype
resin)
Streamline Phenyl
Tosohaas HIC Resins:
TSK Ether 5PW (20 um and 30 um)
TSK Phenyl 5PW (20 um and 30 um)
Phenyl 650S, M, and C
Butyl 650S, M and C
Hexyl-650M and C
Ether-650S and M
Butyl-600M
Super Butyl-550C
PPG-600M
Waters HIC Resins:
[0254] YMC-Pack Octyl Columns-3, 5, 10P, 15 and 25 um with pore
sizes 120, 200, 300 A YMC-Pack Phenyl Columns-3, 5, 10P, 15 and 25
um with pore sizes 120, 200 and 300 A YMC-Pack Butyl Columns-3, 5,
10P, 15 and 25 um with pore sizes 120, 200 and 300 A
CHISSO Corporation HIC Resins:
Cellufine Butyl
Cellufine Octyl
Cellufine Phenyl
JT Baker HIC Resin:
WP HI-Propyl (C3)
Biorad HIC Resins:
[0255] Macroprep t-Butyl Macroprep methyl
Applied Biosystems HIC Resin:
High Density Phenyl--HP2 20 um
[0256] In a further exemplary embodiment, the amount of polypeptide
conjugate loaded onto the HIC medium is between about 0.05 and
about 1.0 mg conjugate/mL resin. In one example, the loaded amount
of polypeptide conjugate is selected between about 0.05 and 0.3 mg
conjugate/mL resin. In another example, the HIC medium is loaded
with between about 0.1 and about 0.2 mg conjugate/mL resin (e.g.,
0.15-0.18 mg/mL). In another embodiment, the amount of polypeptide
conjugate loaded onto the HIC column is optimized for recovery of
peptide conjugate and resolution of glycoforms. In one embodiment,
in which the polypeptide conjugate is EPO-PEG.sub.3, the HIC
loading conditions are selected to create an HIC eluate that
includes less than about 8%, preferably less than about 7%, more
preferably less than about 6%, even more preferably less than about
5% and most preferably less than about 4% of EPO-PEG.sub.2.
[0257] In one embodiment, the loading buffer (the buffer in which
the purified polypeptide conjugate is applied to the HIC column) is
selected to bind the purified polypeptide conjugate to the HIC
medium. Unbound impurities are then washed off the column using a
HIC wash buffer. Consequently, polypeptide conjugates are eluted
using an HIC elution buffer.
[0258] In an exemplary embodiment, the HIC loading buffer, the HIC
wash buffer and the HIC elution buffer each contain one or more
salts, such as sodium acetate (NaOAc), sodium chloride (NaCl),
sodium sulfate (Na.sub.2SO.sub.4) and sodium phosphate. The
concentration ranges for these and other salts are generally
optimized for each type of HIC resin to affect optimal binding of
the polypeptide conjugate being purified.
[0259] In one embodiment, the HIC loading buffer includes sodium
sulfate (Na.sub.2SO.sub.4) or ammonium sulfate,
(NH.sub.4).sub.2SO.sub.4. In an exemplary embodiment, the
concentration of sodium- or ammonium sulfate in the loading buffer
is about 100 mM to about 1200 mM. In another exemplary embodiment,
the concentration of sodium sulfate in the HIC loading buffer is
about 300 mM to about 1100 mM, about 300 mM to about 1000 mM, about
300 mM to about 900 mM, about 300 mM to about 800 mM, about 300 mM
to about 700 mM, about 300 mM to about 600 mM or about 300 mM to
about 500 mM. In yet another embodiment, the concentration of
sodium sulfate in the HIC loading buffer is about 400 mM to about
800 mM. In a further exemplary embodiment, the concentration of
sodium sulfate in the HIC loading buffer is about 500 mM to about
700 mM, and preferably about 600 mM.
[0260] In one embodiment, the HIC loading buffer, HIC wash buffer
and HIC elution buffer include sodium phosphate. In one example,
the concentration of sodium phosphate in any of these buffers is
selected between about 5 mM and about 70 mM. In another example,
the concentration of sodium phosphate in the HIC wash buffer is
selected between about 10 mM and about 50 mM, between about 10 mM
and about 30 mM or between about 20 mM and about 30 mM. In one
particular example, the sodium phosphate concentration in the HIC
wash buffer and elution buffer is about 25 mM.
[0261] In another exemplary embodiment, the HIC wash buffer has a
pH of about 4.0 to about 8.0. In one example, the pH of the HIC
wash buffer is selected from about 5.0 to about 8.0. In another
example, the pH is selected from about 6 to about 8. In yet another
example, the pH is selected from about 6.5 to about 8.0. In a
further embodiment, the pH of the HIC wash buffer is selected from
about 7.0 to about 8.0, from about 7.0 to about 7.9, from about 7.0
to about 7.8, from about 7.0 to about 7.7, from about 7.0 to about
7.6 or from about 7.0 to about 7.5. In one particular example, the
pH of the HIC wash buffer is about 7.5.
[0262] In one embodiment, the purified polypeptide conjugate is
eluted from the HIC resin using a gradient of decreasing sodium
sulfate concentration. Optionally, the elution buffer does not
contain any sodium sulfate.
[0263] In another embodiment, HIC is employed as a method to
separate polypeptide glycoforms, each covalently linked to at least
one poly(alkylene oxide) moiety. In one example, the elution
gradient profile is selected to affect optimal resolution of
different polypeptide glycoforms contained in the purified mixture.
In one embodiment, the HIC elution buffer includes 25 mM sodium
phosphate and a combination of gradient and hold periods spanning a
range of about 600 mM sodium sulfate to no sodium sulfate in the
phosphate buffer is employed to elute polypeptide conjugates from
the HIC medium.
[0264] In an exemplary embodiment, HIC is performed subsequent to
anion exchange chromatography. In one example, the flow-through
from the anion exchanger, which contains the partially purified
polypeptide conjugate is conditioned for hydrophobic interaction
chromatography. In one example, the anion exchange flow-through may
be diluted with a buffer suitable as a loading buffer for HIC. For
example, the anion exchange flow-through can be diluted with a
buffer containing sodium sulfate to adjust the sodium sulfate
concentration in the HIC load (e.g., a sodium sulfate concentration
suitable to bind the polypeptide conjugate to the HIC medium, e.g.,
about 600 mM). Optionally, the anion exchange flow-through is
concentrated before dilution. In another example, the anion
exchange flow-through is subjected to diafiltration/ultrafiltration
for concentration and/or buffer exchange.
III. h) Description of an Exemplary Purification Process
[0265] In one embodiment of the invention, the polypeptide
conjugate of interest is purified from a mixture (e.g., a reaction
mixture, such as a glycoPEGylation reaction) using the exemplary
purification process outlined in FIG. 1. In a first step, the
product of the glycoPEGylation reaction is subjected to anion
exchange chromatography/filtration (e.g., using a Sartobind Q
resin). In one example, impurities are bound by the anion exchange
medium, while the purified polypeptide conjugate is found in the
flow-through. In one embodiment, this anion exchange step is useful
to remove catalytic enzymes used in a glycan remodeling and/or
glycomodification (e.g., glycoPEGylation) reaction performed prior
to the anion exchange procedure. In one example, the anion exchange
step is useful to isolate the polypeptide conjugate from at least
one glycosyltransferase contained in the glycoPEGylation reaction
mixture.
[0266] In a second step, the flow-through of the anion exchange
step containing the partially purified polypeptide conjugate is
conditioned and then loaded onto a hydrophobic interaction
chromatography resin. In one example, the HIC medium is Phenyl
650S.
[0267] In one embodiment, the anion exchange flow-through is
conditioned to generate a HIC loading sample that includes a
sufficient salt concentration to affect binding of the polypeptide
conjugate to the HIC medium. In one embodiment, the anion exchange
flow-trough is diluted with a buffer containing sodium sulfate. In
one embodiment, the dilution buffer contains sufficient sodium
sulfate to generate a HIC loading sample having a sodium sulfate
concentration between about 500 mM and about 700 mM. In one
example, the anion exchange flow-through is diluted so that the HIC
loading sample includes about 600 mM of sodium sulfate. After the
sample is applied to the column, the HIC resin is washed with a
wash buffer to elute unbound impurities. In one example the HIC
wash buffer is a phosphate buffer. In another example, the HIC wash
buffer contains about 25 mM sodium phosphate at a pH of about
7.5.
[0268] Subsequent to washing, the polypeptide conjugate is eluted
from the HIC medium using an elution buffer. In one example, the
polypeptide conjugate is eluted from the HIC medium using a
phosphate buffer (e.g., 25 mM sodium phosphate at pH 7.5) and a
gradient of decreasing sodium sulfate in the phosphate buffer. In
one example, the conjugate is eluted using a gradient from about
600 mM to about 0 mM sodium sulfate. Eluate fractions are collected
and optionally analyzed for product content. Product containing
fractions are pooled and the resulting HIC pool is optionally
conditioned for loading onto a cation exchange medium.
[0269] In one embodiment, the HIC pool is concentrated and the
buffer is exchanged using diafiltration. In one example, the
diafiltration membrane has a MWCO of 10 kDa. In another example,
the volume of the HIC pool is reduced to between about 1/30 and
about 1/10 of the original volume. In a particular example, the HIC
pool volume is reduced to about 1/20 of the original volume. The
buffer may then be exchanged, for example, by diluting the sample
with the new buffer and subsequently re-concentrating the sample.
The dilution and re-concentration steps may be repeated (e.g., 2-6
times) until the new buffer has the desired composition (e.g., the
desired salt conductivity).
[0270] The partially purified polypeptide conjugate may be
transferred into the desired buffer using a two step buffer
exchange. In the first buffer exchange step, the buffer may be
changed to a phosphate buffer that does not include sodium sulfate.
The pH of the resulting conjugate solution may optionally be
adjusted (e.g., using sodium acetate. In a second buffer exchange
step, the buffer may be changed to a buffer system suitable for
loading onto a cation exchanger. For example, the second buffer may
include about 10 mM sodium acetate at a pH of about 5.4. In another
example, the loading buffer for the cation exchange step has a salt
conductivity between about 1.0 and about 3.0 mS/cm (e.g., about 1.5
mS/cm).
[0271] In a third step, the diafiltered HIC pool is subjected to
cation exchange. In one embodiment, the cation exchanger is Source
15S. In one example, the cation exchange medium is useful to
further reduce the content of unwanted glycoforms of the purified
polypeptide conjugate.
[0272] In one example, the partially purified polypeptide conjugate
is applied to the cation exchange medium and unbound impurities are
eluted using a cation exchange wash buffer (e.g., 10 mM sodium
acetate, pH 5.4). The bound polypeptide conjugate is then eluted
using a cation exchange elution buffer. In one embodiment, the
conjugate is eluted using increasing NaCl concentrations in the
above wash buffer. For example, the conjugate is eluted using a
gradient of 0-0.5 M NaCl. In one embodiment, the gradient elution
profile, which may include a combination of gradient and hold
periods, is selected for optimized glycoform resolution. Eluate
fractions are collected and optionally analyzed (e.g., for product
content and purity). Selected product containing fractions are
pooled to form a cation exchange pool.
[0273] In one embodiment, the cation exchange pool is concentrated
and diafiltered into a storage buffer. In one example, this
diafiltration step also uses a 10 kDa MWCO membrane. In one
embodiment, the volume of the cation exchange pool is reduced to
about 1/100 to about 1/25 of its original volume (e.g., about 1/50
of the original volume). The concentrated cation exchange pool is
then subjected to buffer exchange, for example, by diluting the
sample with the new buffer and subsequently re-concentrating the
sample. The dilution and re-concentration step may be repeated
(e.g., 2-6 times) until the new buffer has the desired composition.
The final retentate is reconstituted into a storage buffer.
Exemplary storage buffers include those having a sodium chloride
concentration that is in the physiological range. For example, the
storage buffer may be a sodium acetate buffer including about 150
mM NaCl. The concentrated product pool is reconstituted in the
storage buffer to reach a desired peptide concentration. In one
embodiment, the final conjugate concentration is selected between
about 0.5 and about 2 mg/mL. The final solution is optionally
sterile filtered, for example through a cellulose acetate
membrane.
[0274] The purification process outlined in FIG. 1, may optionally
include an additional chromatography step. In one embodiment, the
process includes a hydroxyapatite (HA) or fluoroapatite
chromatography step. In one example, the apatite chromatography is
performed after anion exchange chromatography. In another example,
the apatite chromatography is performed after HIC. In yet another
example, the apatite chromatography is performed after cation
exchange chromatography. The partially purified polypeptide
conjugate solution may be desalted, for example, using a size
exclusion column (e.g. G25) to lower the salt conductivity of the
conjugate solution in preparation for apatite chromatography.
[0275] In an exemplary embodiment, the polypeptide conjugate
purified by the above described process is an EPO-conjugate.
II. Compositions
[0276] In another aspect the invention provides a composition made
by a method of the invention. In one embodiment, the invention
provides a composition including a first polypeptide conjugate,
said first polypeptide conjugate having a first number of
poly(alkylene oxide) moieties, each of the poly(alkylene oxide)
moieties covalently linked to the first polypeptide via an intact
glycosyl linking group. The composition is made by a method
including: (a) contacting a mixture comprising the first
polypeptide conjugate with a hydrophobic interaction chromatography
(HIC) medium; and (b) eluting the first polypeptide conjugate from
the hydrophobic interaction chromatography medium.
[0277] In another aspect, the invention provides an isolated first
polypeptide conjugate made by a method comprising: separating the
first polypeptide conjugate including a first number of
poly(alkylene oxide) moieties covalently linked to a first
polypeptide, from a second polypeptide conjugate comprising a
second number of poly(alkylene oxide) moieties covalently linked to
a second polypeptide. In one embodiment, the first number is
selected from 1 to 20 and the second number is selected from 0-20.
In another embodiment, the first number and the second number are
different. The two polypeptide conjugates are separated by: (a)
contacting a mixture comprising the first polypeptide conjugate and
the second polypeptide conjugate with a hydrophobic interaction
chromatography (HIC) medium; and (b) eluting the first polypeptide
conjugate from the hydrophobic interaction chromatography
medium.
[0278] In one example, according to any of the above embodiments,
the first polypeptide is a member selected from erythropoietin
(EPO), bone morphogenetic protein 2 (BMP-2), bone morphogenetic
protein 7 (BMP-7), bone morphogenetic protein 15 (BMP-15),
neurotrophin-3 (NT-3), von Willebrand factor (vWF) protease,
granulocyte colony stimulating factor (G-CSF),
granulocyte-macrophage colony stimulating factor (GM-CSF),
interferon alpha, interferon beta, interferon gamma,
.alpha..sub.1-antitrypsin (.alpha.-1 protease inhibitor),
glucocerebrosidase, tissue-type plasminogen activator (TPA),
interleukin-2 (IL-2), leptin, hirudin, urokinase, human DNase,
insulin, hepatitis B surface protein (HbsAg), chimeric diphtheria
toxin-IL-2, human growth hormone (hGH), human chorionic
gonadotropin (hCG), thyroid peroxidase (TPO), alpha-galactosidase,
alpha-L-iduronidase, beta-glucosidase, alpha-galactosidase A, acid
.alpha.-glucosidase (acid maltase), anti-thrombin III (AT III),
follicle stimulating hormone (FSH), glucagon-like peptide-1
(GLP-1), glucagon-like peptide-2 (GLP-2), fibroblast growth factor
7 (FGF-7), fibroblast growth factor 21 (FGF-21), fibroblast growth
factor 23 (FGF-23), Factor X, Factor XIII, prokinetisin,
extendin-4, CD4, tumor necrosis factor receptor (TNF-R),
.alpha.-CD.sub.20, P-selectin glycoprotein ligand-1 (PSGL-1),
complement, transferrin, glycosylation-dependent cell adhesion
molecule (GlyCAM), neural-cell adhesion molecule (N-CAM), TNF
receptor-IgG Fc region fusion protein, anti-HER2 monoclonal
antibody, monoclonal antibody to respiratory syncytial virus,
monoclonal antibody to protein F of respiratory syncytial virus,
monoclonal antibody to TNF-.alpha., monoclonal antibody to
glycoprotein IIb/IIIa, monoclonal antibody to CD20, monoclonal
antibody to VEGF-A, monoclonal antibody to PSGL-1, monoclonal
antibody to CD4, monoclonal antibody to a-CD3, monoclonal antibody
to EGF, monoclonal antibody to carcinoembryonic antigen (CEA) and
monoclonal antibody to IL-2 receptor. Mutant forms of any of the
above polypeptides are also within the scope of the invention.
[0279] In yet another aspect, the invention provides a composition
including a first erythropoietin (EPO) conjugate, the first EPO
conjugate having a first number of poly(alkylene oxide) moieties
covalently linked to an EPO polypeptide via a glycosyl linking
group (e.g., an intact glycosyl linking group). The composition is
made by a method including: (a) contacting a mixture including the
first EPO conjugate with an anion exchange medium; (b) eluting the
first EPO conjugate from the anion exchange medium, forming a first
eluate comprising the first EPO conjugate; (c) contacting the first
eluate with a hydrophobic interaction chromatography (HIC) medium;
and (d) eluting the first EPO conjugate from the hydrophobic
interaction chromatography medium.
[0280] In a further aspect, the invention provides a pharmaceutical
formulation including a composition made by a method of the
invention and a pharmaceutically acceptable carrier. In one
embodiment, the pharmaceutical formulation includes an isolated
polypeptide conjugate made by a method of the invention and a
pharmaceutically acceptable carrier. In one example according to
this embodiment, the isolated polypeptide conjugate is an
EPO-conjugate.
III. Methods of Treatment
[0281] In another aspect, the invention provides methods of
treatment utilizing a composition (e.g., an isolated polypeptide
conjugate) made by a method of the invention. In one embodiment,
the invention provides a method of treating a condition in a
subject in need thereof, the condition characterized by compromised
red blood cell production in the subject, the method comprising:
administering to the subject an amount of a composition of the
invention, effective to ameliorate the condition in the subject. In
one example, the subject is a mammal, such as a human. In another
example, the composition includes an EPO conjugate made by a method
of the invention.
[0282] In another embodiment, the invention provides a method of
treating a tissue injury in a subject in need thereof. In one
example, the injury is caused by a member selected from ischemia,
trauma, inflammation and contact with a toxic substance. The method
includes: administering to a subject an amount of a composition
made by a method of the invention (e.g., an isolated polypeptide
conjugate) that is effective in ameliorating the damage associated
with the tissue injury. In one example, the subject is a mammal,
such as a human. In another example, the composition includes an
EPO conjugate made by a method of the invention.
[0283] In another embodiment, the invention provides a method of
enhancing red blood cell production in a mammal, said method
comprising administering to said mammal a composition made by a
method of the invention. In one example, the subject is a mammal,
such as a human. In another example, the composition includes an
EPO conjugate made by a method of the invention.
[0284] The following examples are provided to illustrate the
methods of the present invention, but not to limit the claimed
invention.
EXAMPLES
Analytical Methods
Protein Concentration Determination
[0285] The protein concentration was determined using either a UV
method (280 nm) or was determined using BCA Protein Assay kit
according to manufacture's instructions (Pierce).
Conductivity Measurement
[0286] The conductivity of process samples and buffers was measured
using a conductivity probe (VWR 2052) according to the
manufacturer's instructions.
EPO-PEG Purity and PEG State Assay (C3 RP-HPLC)
[0287] The ratio of each EPO-PEG form [EPO-(SA-PEG-10 kDa).sub.1-4]
was determined using C.sub.3 RP HPLC chromatography (Zorbax
300SB-C3, 150.times.2.1 mm, 5 micron, 45.degree. C.). The HPLC was
performed using the following solutions: A, 0.1% TFA in water, and
B, 0.09% TFA in ACN. The mobile phase was performed as a gradient
from 42-55% B over 14 min, 55-95% B over 2 min, a 2 min wash at 95%
B and then 95-42% B over 2 min. The total time conduct the
chromatography was 30 min using a flow rate of 0.6 mL/min. Mixture
1 was used as a reference standard to test system suitability. The
injection volume was varied to give a standard injection
concentration of 5 to 10 .mu.g of EPO-(SA-PEG-10 kDa).sub.3 based
on protein. Protein absorbance was detected at 214 nm and the peak
areas of the EPO species were used to determine the protein purity.
All peaks were integrated using 32 karat software. The
EPO-(SA-PEG-10 kDa).sub.4 peak could typically not be integrated
accurately due to its small area.
Protein Purity and Aggregation by SEC HPLC.
[0288] The EPO-(SA-PEG-10 kDa).sub.3 isoform purity and aggregation
were determined by SEC HPLC chromatography (TSK-gel
G5000PW.times.L, 7.8.times.300 mm, 10 micron, 4.degree. C.). The
isocratic mobile phase (100 mM sodium phosphate, 150 mM sodium
chloride, pH 7.0) was used to perform the method at a flow rate of
0.5 mL/min.
Example 1
Development of a Purification Process for the Purification of
GlycoPEGylated EPO Using Hydrophobic Interaction Chromatography
[0289] This example describes the development of an isolation
process for the isolation of EPO conjugates from a glycoPEGylation
reaction mixture. The resulting process is characterized by high
overall EPO conjugate recovery and produces the desired EPO
conjugate [EPO-(SA-PEG-10 kDa).sub.3] in high purity. The
composition produced by the process is essentially free of other
EPO-PEG glycoforms, such as mono-, di- and tetra-PEGylated EPO
conjugates.
[0290] The desired EPO-PEG conjugate is a glycoPEGylated
erythropoietin protein that contains three 10 kDa mPEG groups
attached to each of the three monoantennary N-linked glycans. The
EPO polypeptide is produced by expression of the protein from Sf9
cells using a Baculovirus infection protocol. The insect cell
expression system produces EPO with three N-linked glycans, at
Asn.sup.24, Asn.sup.38 and Asn.sup.83, each containing a
trimannosyl core as the predominant species. A variety of other
glycan structures are present in small amounts that vary with
fermentation conditions. The other structures include trimannosyl
core with an additional GlcNAc, higher mannose forms (Man.sub.4,
Man.sub.5), missing glycans, and GlcNAc-(Fuc) stub arising as a
result of an endoglycosidase-type (Endo-H) activity. A small
percentage of the EPO molecules contain O-linked glycans at
Ser.sup.126. A very low level (<1%) of phosphorylcholine-linked
glycans (PC-glycan) has also been identified in the insect
cell-derived EPO. Upon GlycoPEGylation with MBP-GnT1, MBP-GalT1,
and MBP-ST3Gal3 or ST3Gal3 with CMP-SA-PEG-10 kDa, the predominant
product is EPO-(SA-PEG-10 kDa).sub.3 which contains three PEGylated
mono-antennary N-linked glycans. EPO-(SA-PEG-10 kDa).sub.1-2 are
also present in lesser amounts, arising from the EPO forms missing
one or more glycans or containing one or more GlcNAc-stub glycans.
EPO-(SA-PEG-10 kDa).sub.4 (and higher) are produced at very low
levels. These higher PEGylated species are thought to result from
the GlycoPEGylation of the EPO glycoforms which contain the
tri-mannosyl core with an additional GlcNAc on the Man.alpha.1,6
branch. Both branches of these glycans can be built out and
PEGylated under the reaction conditions, resulting in biantennary
PEGylated glycans on a tetra-PEG (or higher) EPO species. Exemplary
EPO glycoforms are depicted in FIG. 3.
[0291] GlycoPEGylated EPO isolated using reversed-phase
chromatography and cation exchange chromatography (e.g., on
SP-Sepharose HP) provided a composition containing greater than or
equal to about 85% EPO-(SA-PEG-10 kDa).sub.3, about 3-14%
EPO-(SA-PEG-10 kDa).sub.2, about 1-8% EPO-(SA-PEG-10 kDa).sub.4-6
and less than or equal to about 1% EPO-(SA-PEG-10 kDa).sub.i. In
the following, the above composition is referred to as Mixture
1.
[0292] Efforts were undertaken to replace the reverse phase
chromatography step with another chromatography step, which does
not employ organic solvents but is capable of resolving EPO
glycoforms. Large amounts of organic solvents are associated with
environmental concerns and may not be used in certain facilities
processing biologic drug products. In addition, the stability of
EPO-PEG conjugates, such as EPO-(SA-PEG-10 kDa).sub.3 in solutions
with a high concentration of organic solvents (e.g., acetonitrile)
is a concern. Hydrophobic Interaction Chromatography (HIC) was
investigated as a potential replacement for the reverse phase
chromatography step.
[0293] It was discovered that HIC was capable of separating
different PEG states (glycoforms) contained in a mixture, which
results from a glycoPEGylation process (e.g., fractionation of
isoforms EPO-(SA-PEG-10 kDa).sub.1-4). The HIC purification method
was optimized by evaluating a variety of HIC resins and process
parameters. A process based on Phenyl 650S resin (e.g., Tosohaas,
Toyopearl) was selected for incorporation into the new isolation
process.
[0294] Also incorporated into the new process was an anion exchange
step, which is useful to remove enzyme components of the
glycoPEGylation reaction. Enzymes are bound by the anion exchange
medium, while the EPO-PEG conjugates EPO-(SA-PEG-10 kDa).sub.1-6
are found in the flow through.
1.1. Methods
Hydrophobic Interaction Chromatography Conditions (Initial Resin
Screen)
[0295] Initial HIC resin screening experiments were performed using
Tricorn 5 columns packed to 5 cm bead heights. Selected hydrophobic
interaction chromatography resins are summarized in Table 1,
below.
TABLE-US-00002 TABLE 1 Summary of Evaluated HIC Resins and Elution
Conditions Resin Buffer A Buffer B HIC Resins Set I: A Ether-5PW 30
mM Na phosphate, pH 6.5 Buffer A + 1 M NaCl, pH 6.5 B Butyl-S-FF 25
mM Na phosphate, pH 7.0 Buffer A + 1 M (NH.sub.4).sub.2SO.sub.4, pH
7.0 C Butyl-FF 25 mM Na phosphate, pH 7.0 Buffer A + 1 M
(NH.sub.4).sub.2SO.sub.4, pH 7.0 D Octyl-FF 25 mM Na phosphate, pH
7.0 Buffer A + 1 M (NH.sub.4).sub.2SO.sub.4, pH 7.0 E Phenyl-FF Low
sub 25 mM Na phosphate, pH 7.0 Buffer A + 1 M
(NH.sub.4).sub.2SO.sub.4, pH 7.0 F Phenyl-FF High sub 25 mM Na
phosphate, pH 7.0 Buffer A + 1 M (NH.sub.4).sub.2SO.sub.4, pH 7.0
HIC Resin Set II: G Butyl 650M 25 mM Na phosphate, pH 7.0 Buffer A
+ 1 M (NH.sub.4).sub.2SO.sub.4, pH 7.0 H Phenyl 650M 25 mM Na
phosphate, pH 7.0 Buffer A + 4 M NaCl, pH 7.0 I Phenyl 650M 25 mM
Na phosphate, pH 7.0 Buffer A + 1 M (NH.sub.4).sub.2SO.sub.4, pH
7.0 J Phenyl-FF Low sub 25 mM Na phosphate, pH 7.0 Buffer A + 4 M
NaCl, pH 7.0 K Phenyl-FF Low sub 25 mM Na phosphate, pH 7.0 Buffer
A + 1 M (NH.sub.4).sub.2SO.sub.4, pH 7.0 HIC Resin Set III: L
Phenyl 650M 20 mM NaOAc, pH 5.0 Buffer A + 1 M Na.sub.2SO.sub.4 +
0.5 M NaCl, pH 5.0 M Phenyl 650M 20 mM NaOAc, pH 5.0 Buffer A + 1 M
Na.sub.2SO.sub.4, pH 5.0 N Phenyl 650M 25 mM Na phosphate, pH 7.0
Buffer A + 1 M Na.sub.2SO.sub.4, pH 7.0 O Phenyl 650M 25 mM Na
phosphate, 20% Buffer A + 4 M NaCl, pH 7.0 ethylene glycol, pH 7.0
P Phenyl 650M 25 mM Na phosphate, pH 7.0 Buffer A + 4 M NaCl, pH
7.0 Q Phenyl 650M 25 mM Na phosphate, pH 7.0 Buffer A + 1 M
(NH.sub.4).sub.2SO.sub.4, pH 7.0
[0296] A Tricorn 5 column packed with 1 mL (0.5 cm.times.5 cm) of
the appropriate resin (Table 1) or a prepacked HiTrap column (1 mL,
0.7 cm.times.2.5 cm) was attached to an AKTA FPLC system (GE
Healthcare). Product elution was monitored by absorbance at 280 nm.
Each column was equilibrated with 5 column volumes (CV) of Buffer B
(as indicated in Table 1). Mixture 1 (100 mcg, 250 mcL) was diluted
to 1.25 mL with 1 mL of Buffer B and injected using a 2 mL sample
loop. The unbound material was washed with 5 CV of Buffer B. The
EPO-(SA-PEG-10 kDa).sub.3 was eluted with the following gradient
elution using Buffer A (as indicated in Table 1): 100-0% Buffer B
over 20 CV followed by 0% Buffer B for 5 CV. All steps were
performed at 0.64 mL/min (196 cm/hr). The resulting chromatograms
were compared and selected fractions were analysed by SDS-PAGE.
Resins were selected for their capability to retain the EPO-PEG
conjugates, their capability to resolve different glycoforms and
peak shapes.
1.2. Results
[0297] The following Hydrophobic Interaction Chromatography (HIC)
resins; Butyl-FF, Butyl-S--FF, Octyl-FF, Phenyl-FF High sub and
Phenyl-FF Low sub were initially tested by injecting Mixture 1 in
buffer B: 25 mM Na phosphate, 1 M ammonium Sulfate, pH 7.0. Under
the conditions tested, the Phenyl-FF Low sub appeared to be the
best resin for the binding and elution of Mixture 1. Mixture 1 was
not bound by Butyl-S--FF. Butyl-FF, Octyl-FF, and Phenyl-FF High
sub bound Mixture 1 fairly tightly and the elution peaks were
extremely broad (.about.15 CV).
[0298] In a second round of HIC investigation, Phenyl-FF Low sub
was compared to Phenyl 650M and Butyl 650M using either 25 mM Na
phosphate, 1 M ammonium Sulfate, pH 7.0 or 25 mM Na phosphate, 4 M
ammonium Sulfate, pH 7.0 as binding buffer (loading buffer). Butyl
650M was only tested with ammonium sulfate and resulted in a very
wide and tailing elution peak. Both Phenyl 650M and Phenyl-FF low
sub had a more symmetric peak shape when using sodium sulfate. The
use of sodium chloride resulted in extremely broad elution
peaks.
[0299] A third round of HIC testing, compares the Phenyl 650M
purification of Mixture 1 with different buffers. Buffer B: 1 M Na
Sulfate+0.5 M NaCl+20 mM NaOAc, pH 5.0 was tested as this condition
had worked for purification of the EPO intermediate prior to
PEGylation. This condition resulted in a broad elution peak with
multiply bumps, as did the same buffer without 0.5M NaCl (1 M Na
Sulfate+20 mM NaOAc, pH 5.0). Phenyl 650M was tested using Mixture
1 bound to the column using 4 M NaCl+buffer A, pH 7.0 and the
elution was performed with 25 mM Na phosphate, 20% ethylene glycol,
pH 7.0. The addition of ethylene glycol did not significantly
improve the broad elution profile resulting from NaCl in buffer B.
Hence, conditions N and Q were found to be superior to other tested
conditions. Sodium sulfate, which is preferred in manufacturing
gave an elution profile as good or better that the ammonium
sulfate.
Optimization of Chromatography Conditions (HIC)
[0300] Elution of Glycosyltransferases from Tosohaas Phenyl 650 M
Resin
[0301] A Tricorn 5 column packed with a 1 mL (0.5 cm.times.5 cm)
Phenyl 650M resin was attached to an AKTA FPLC system continuously
monitoring absorbance at A280. The column was equilibrated with 5
column volumes (CV) of Buffer B (1 M sodium sulfate, 25 mM sodium
phosphate, pH 7.0). Product elution was monitored by absorbance at
280 nm.
[0302] Four separate sample injections of the following materials
were applied to the column using a 2 mL sample loop: 1. Mixture 1
(100 mcg, 250 mcL) diluted to 1.25 mL with 1 mL of Buffer B. 2.
MBP-SBD-ST3Gal3 (98 mcL, 98 mcg) diluted to 1.225 mL with 1.127 mL
of Buffer B. 3. MBP-GnT1 (196 mcL, 57.4 mcg) diluted to 1.225 mL
with 1.029 mL of Buffer B. 4. MBP-GalT1 (196 mcL, 186 mcg) diluted
to 1.225 mL with 1.029 mL Buffer B.
[0303] After each sample was injected, any unbound material was
washed from the column using 5 CV of Buffer B. Any bound material
was eluted from the column using a gradient using Buffer A (25 mM
sodium phosphate, pH 7.0): 100-0% Buffer B over 20 CV followed by
0% Buffer B for 5 CV (Table 1, Set III, N). Chromatography
operations were performed at a flow rate of 0.64 mL/min. Elution
profiles were compared and selected fractions were analysed by
SDS-PAGE.
[0304] The glycoPEGylation enzymes were individually injected on
Phenyl 650M to compare the elutio profiles to Mixture 1 using the
following condition: Buffer A: 25 mM Na phosphate, pH 7.0. Buffer
B: 1 M Na Sulfate+buffer A, pH 7.0. Elution gradient: 100-0% B over
20 cv. The MBP-SBD-ST3Gal3, MBP-GnT1 and MBP-GalT1 all eluted in
the same portion of the gradient. Mixture 1 elutes just prior to
the enzymes. Although Mixture 1 and glycoPEGylation enzymes do not
completely co-elute, Mixture 1 and the leading portion of the
enzyme elution peaks seemed to overlap.
Optimization of the Sodium Sulfate Concentration using Phenyl 650M
Chromatography
[0305] The concentration of sodium sulfate required to bind Mixture
1 bind Phenyl 650M was investigated. A Tricorn 5 column was packed
with Phenyl 650M resin (1 mL, 0.5 cm 5 cm) as described above and
attached to a Varian HPLC system. Each column was equilibrated with
5 column volumes (CV) of one of the buffers listed below. Product
elution was monitored by absorbance at 280 nm.
[0306] Five separate injections of Mixture 1 (100 mcg, 250 mcL
diluted to 1.25 mL with 1 mL Buffer B) were applied to the column
using a 2 mL sample loop. Each chromatography used one of the
following sodium sulfate concentrations in Buffer B: 1. 1.0 M
sodium sulfate, 25 mM sodium phosphate, pH 7.5; 2. 0.8 M sodium
sulfate, 25 mM sodium phosphate, pH 7.5; 3. 0.6 M sodium sulfate,
25 mM sodium phosphate, pH 7.5; 4. 0.4 M sodium sulfate, 25 mM
sodium phosphate, pH 7.5; 5. 0.3 M sodium sulfate, 25 mM sodium
phosphate, pH 7.5.
[0307] Any unbound material was washed from the column with 5 CV of
Buffer B. EPO-(SA-PEG-10 kDa).sub.3 was eluted using a gradient of
Buffer A (25 mM sodium phosphate, pH 7.0): 100-0% Buffer B over 20
CV (gradient change 5% Buffer B/CV), and then 0% Buffer B for 5 CV.
Chromatography operations were performed at a flow rate of 0.64
mL/min (196 cm/hr). EPO-(SA-PEG-10 kDa).sub.3 peak fractions were
stored at 4.degree. C. until analysis by SDS-PAGE.
[0308] A minimum of 0.4 M sodium sulfate was required to bind
Mixture 1 to the column. As expected the higher the sodium sulfate
concentration the greater the retention time on the column. The
same gradient slope (change in % B per minute) was maintained for
all five purification runs. As the initial concentration of buffer
B was reduced, the actual gradient (change in salt
concentration/minute) became more shallow. This results in the
increase in elution peak width seen as the buffer B start
concentration decreases.
Optimization of the Sodium Sulfate Elution Gradient using Phenyl
650S Chromatography
[0309] A Tricorn 5 column was packed with Phenyl 650S resin (1 mL,
0.5 cm.times.5 cm) as described above and attached to an AKTA FPLC
system. Product elution was monitored by absorbance at 280 nm.
Three separate purifications were performed each using a different
elution gradient. The columns from each experiment were
equilibrated with 5 column volumes (CV) of Buffer B (25 mM sodium
phosphate, 0.6 M sodium sulfate, pH 7.5).
[0310] Mixture 1 (100 mcg, 250 mcL) was diluted to 1.25 mL with 1
mL Buffer B and injected onto the column using a 2 mL sample loop.
Any unbound material was washed from the column using 5 CV of
Buffer B. The EPO-(SA-PEG-10 kDa).sub.3 product was eluted from the
column using one of the following gradients using Buffer A (25 mM
sodium phosphate, pH 7.5). Gradient 1: 100-0% Buffer B over 20 CV
(gradient change 5% Buffer B/CV) followed by 0% Buffer B for 5 CV.
Gradient 2: 100-0% Buffer B over 12 CV (gradient change 8.33%
Buffer B/CV) followed by 0% Buffer B for 5 CV. Gradient 3: 100-60%
Buffer B over 2 CV, hold for 1 CV, 60-20% Buffer B over 20 CV
(gradient change 2% Buffer B/CV), 20-0% Buffer B over 2 CV and then
0% Buffer B for 5 CV. Chromatography operations were performed at a
flow rate of 0.64 mL/min. Chromatography elution profiles were
compared and selected fractions were analyzed by SDS PAGE. Out of
the three gradient profiles tested, Gradient 3 provided the best
separation of the EPO-PEG species.
Dynamic Range of the Process Parameters (Tosohaas Phenyl 650 S)
Effect of pH on the Separation of EPO-PEGS
[0311] The effect of pH on the ability of the Phenyl 650S
chromatography process to separate the PEG states of EPO-PEG was
examined. A Tricorn 10 column was packed with 15.7 mL (1.0 cm
id.times.20 cm) of Phenyl 650S. The column was equilibrated with 5
column volumes (CV) of buffer B (25 mM sodium phosphate, 0.6 M
sodium sulfate, selected pH). In separate experiments, the process
was repeated except the buffer pH was 6.5, 7.0, 7.5 or 8.0 in both
Buffer A and B. The EPO-PEG.sub.x mixture (0.8 mg EPO protein
conjugate; 1 mL) solution was adjusted to a sodium sulfate
concentration of 0.6 M by addition of 0.4 mL of Buffer A (25 mM
sodium phosphate, selected pH) and 0.6 mL of buffer (2 M sodium
sulfate, 25 mM sodium phosphate, selected pH). This solution was
then diluted with an equal volume of Buffer B and the entire sample
(4 mL) was injected onto the column. The column was washed with 5
CV of Buffer B and the product eluted using a gradient using Buffer
A. A gradient of 100-60% Buffer B over 2 CV, hold for 1 CV, 60-35%
Buffer B over 13 CV, hold for 1 CV and then 0% Buffer B for 5 CV.
The chromatography flow rate was 2.0 mL/min (150 cm/hr) and the
column elution monitored by the absorbance at 280 nm. Fractions
(1.6 mL) were collected in Nunc 96-well Microtiter plates. The
chromatography elution profiles were compared and selected
fractions were analyzed by SDS PAGE. Analysis of the product pool
is summarized in Table 2, below:
TABLE-US-00003 TABLE 2 Effect of pH on Separation and Recoveries of
EPO-PEGs using Phenyl 650S.sup.1 Step MBP.sup.6 Buffer Yield.sup.3
EPO-PEG.sub.3.sup.4 EPO-PEG.sub.2.sup.5 [mcg/mg Aggregate.sup.7
pH.sup.2 [%] [%] [%] EPO] [%] pH 6.5 60.3 95.1 4.9 <0.085 0 pH
7.0 66.7 96.3 3.7 <0.077 0 pH 7.5 61.6 97.5 2.5 <0.083 0 pH
8.0 65.4 96.6 3.4 <0.078 0 .sup.1EPO-PEG (85.1% EPO-PEG.sub.3
and 14.9% EPO-PEG.sub.2 (Table 7). .sup.2The pH of both, Buffer A
and B. .sup.3The step yield was calculated as the ratio of EPO-PEG
protein recovered after HIC chromatography (combined fractions)
versus the EPO-PEG injected onto the column .sup.4The percent of
EPO-(SA-PEG-10 kDa).sub.3 in the main product peak. .sup.5The
percent of EPO-(SA-PEG-10 kDa).sub.2 in the main product peak.
.sup.6The amount of MBP protein in the main product peak as
determine by ELISA versus the amount of EPO-PEG protein. .sup.7The
amount of aggregate EPO-PEG in the product peak observed by SEC
[0312] Standard purification conditions (Buffer A: 25 mM sodium
phosphate, pH 7.5. Buffer B: 25 mM sodium phosphate, 0.6M sodium
sulfate, pH 7.5 (with 0.05 mg EPO-(SA-PEG-10 kDa).sub.1-4/mL resin
loaded) were compared to purification runs with buffer A and B
prepared at pH 6.5, 7.0 and 8.0. The elution peaks (A280) are
shifted slightly to the left and right of the standard elution
condition. The X-axis of these A280 traces were shifted to overlay
the peaks. Comparison of the X-axis shifted peaks shows similar
peak traces with respect to the elution of EPO-(SA-PEG-10
kDa).sub.2, EPO-(SA-PEG-10 kDa).sub.3, and EPO-(SA-PEG-10
kDa).sub.4. This is especially true for pH 7.0, 7.5 and 8.0. The pH
6.5 run had a slightly lower peak max and a little less resolution
was noted between the EPO-(SA-PEG-10 kDa).sub.3 and EPO-(SA-PEG-10
kDa).sub.4 peaks.
[0313] Table 2, above, compares EPO-(SA-PEG-10 kDa).sub.3 recovery,
MBP-ELISA, RP-HPLC, and SEC analysis data. No significant
difference was observed between the EPO-(SA-PEG-10 kDa).sub.3
purified within the 6.5 to 8.0 pH range. Recovery is between 60.3
and 66.7% (BCA analysis). MBP-ELISA indicates <0.077-0.085 mcg
MBP/mg EPO-(SA-PEG-10 kDa).sub.3 remaining RP-HPLC analysis show
that the amount of EPO-(SA-PEG-10 kDa).sub.2 remaining in the
EPO-(SA-PEG-10 kDa).sub.3 peak is between 2.5 and 4.9%. Integration
of EPO-(SA-PEG-10 kDa).sub.4 is difficult at low levels but is
detected by silver stain SDS-PAGE. No aggregation of EPO-(SA-PEG-10
kDa).sub.3 is detected by SEC analysis. SDS-PAGE analysis shows
that the purity of each EPO-(SA-PEG-10 kDa).sub.3 pool is similar.
However, the pH 6.5 purified material contained a higher amount of
a approximate 50 kDa proteolyses band. A minor amount of this
proteolysis product was also detected for the pH 7.0 purified
material.
Effect of Sodium Phosphate Concentration on the Separation of
EPO-PEGs
[0314] The effect of sodium phosphate concentration on the ability
of the Phenyl 650S chromatography process to separate the PEG
states of EPO-PEG was examined. A Tricorn 10 column was packed with
15.7 mL (1.0 cm id.times.20 cm) of Phenyl 650S. The column was
equilibrated with 5 column volumes (CV) of buffer B (various
concentrations of sodium phosphate, 0.6 M sodium sulfate, pH 7.5).
In separate experiments, the process was repeated except the sodium
phosphate concentration was 15, 25 or 50 mM in both Buffer A and B.
The EPO-PEG.sub.x mixture (0.8 mg EPO protein; 1 mL) solution was
adjusted to a sodium sulfate concentration of 0.6 M by addition of
0.4 mL of Buffer A (various concentrations of sodium phosphate, pH
7.5) and 0.6 mL of buffer (2 M sodium sulfate, various
concentrations of sodium phosphate, pH 7.5). This solution was then
diluted with an equal volume of Buffer B and the entire sample (4
mL) was injected onto the column. The column was washed with 5 CV
of Buffer B and the product eluted using a gradient using Buffer A.
A gradient of 100-60% Buffer B over 2 CV, hold for 1 CV, 60-35%
Buffer B over 13 CV, hold for 1 CV and then 0% Buffer B for 5 CV.
The chromatography flow rate was 2.0 mL/min (150 cm/hr) and the
column elution monitored by the absorbance at 280 nm. The
chromatography elution profiles were compared and selected
fractions were analyzed by SDS PAGE. The pooled fractions of the
major product peak were analyzed. Results are summarized in Table
3, below:
TABLE-US-00004 TABLE 3 Effect of Sodium Phosphate Concentration on
Separation and Recovery of EPO-PEGs using Phenyl 650S..sup.1 Sodium
Step EPO- EPO- MBP.sup.6 Phosphate Yield.sup.3 PEG.sub.3.sup.4
PEG.sub.2.sup.5 [mcg/mg Aggregate.sup.7 Concentration.sup.2 [%] [%]
[%] EPO] [%] 15 mM 61.5 95.5 4.5 <0.083 0 25 mM 61.9 96.3 3.7
<0.085 0.22 50 mM 60.2 95.8 4.2 <0.085 0.34 .sup.1EPO-PEG
(85.1% EPO-PEG.sub.3 and 14.9% EPO-PEG.sub.2 (Table 7). .sup.2The
sodium phosphate concentration of both, Buffer A and B. .sup.3The
step yield was calculated as the ratio of EPO-PEG protein recovered
after HIC chromatography (combined fractions) versus the amount
EPO-PEG injected onto the column .sup.4The percent of
EPO-(SA-PEG-10 kDa).sub.3 in the main product peak. .sup.5The
percent of EPO-(SA-PEG-10 kDa).sub.2 in the main product peak.
.sup.6The amount of MBP protein in the main product peak as
determine by ELISA assay versus the amount of EPO-PEG protein.
.sup.7The amount of aggregate EPO-PEG in the product peak observed
by SEC.
[0315] Standard purification conditions (Buffer A: 25 mM sodium
phosphate, pH 7.5. Buffer B: 25 mM sodium phosphate, 0.6M sodium
sulfate, pH 7.5 (0.05 mg EPO-(SA-PEG-10 kDa).sub.1-4/mL resin
loaded) were compared to purification runs with buffer A and B
prepared with 15 and 50 mM sodium phosphate. Resulting A280 elution
profiles were compared. The 15 mM Na phosphate eluted material
elutes earliest, followed by the 25 mM Na phosphate and finally 50
mM Na phosphate. The shift in elution profile is due to the
relative changes in buffer conductivity (see table 3). The X-axis
of these A280 traces were shifted to overlay the peaks. Comparison
of the X-axis shifted peaks shows similar peak traces with respect
to the elution of EPO-(SA-PEG-10 kDa).sub.2, EPO-(SA-PEG-10
kDa).sub.3, and EPO-(SA-PEG-10 kDa).sub.4.
[0316] Table 3 compares EPO-(SA-PEG-10 kDa).sub.3 recovery,
MBP-ELISA, RP-HPLC, and SEC analysis data. No significant
difference was observed between the EPO-(SA-PEG-10 kDa).sub.3
purified within this 15 to 50 mM sodium phosphate concentration
range. Recovery is between 60.2 and 61.9%. MBP-ELISA indicates
<0.083-0.085 mcg MBP/mg EPO-(SA-PEG-10 kDa).sub.3 remaining
RP-HPLC analysis showed that the amount of EPO-(SA-PEG-10
kDa).sub.2 remaining in the EPO-(SA-PEG-10 kDa).sub.3 peak is
between 3.7 and 4.5%. Integration of the EPO-(SA-PEG-10 kDa).sub.4
peak was difficult at low levels but is detected by silver stain
SDS-PAGE. SDS-PAGE analysis showed that the purity of each
EPO-(SA-PEG-10 kDa).sub.3 pool is comparable as determined by
colloidal blue and silver stained gels. No notable differences in
purity were detected by SDS-PAGE.
Effect of Sodium Sulfate Concentration on the Separation of
EPO-PEGs
[0317] The robustness and the capability of the Phenyl 650S
chromatography step to separate the PEG states of EPO-PEG was
examined with respect to variations in the Na sulfate
concentrations in buffer B. A Tricorn 10 column was packed with
15.7 mL (1.0 cm id.times.20 cm) of Phenyl 650S. The column was
equilibrated with 5 column volumes (CV) of buffer B (25 mM sodium
phosphate, various concentrations of sodium sulfate, pH 7.5). In
separate experiments, the process was repeated except the buffer
sodium sulfate concentrations of 0.5, 0.6 and 0.7 M of Buffer B
were varied. The EPO-PEG.sub.x mixture (0.8 mg EPO protein; 1 mL)
solution was adjusted to a sodium sulfate concentration by the
addition of 0.3-0.5 mL of Buffer A (25 mM sodium phosphate, pH 7.5)
and 0.5-0.7 mL of buffer (2 M sodium sulfate, 25 mM sodium
phosphate, pH 7.5) depending on the sodium sulfate concentration
used in the experiment. This solution was then diluted with an
equal volume of Buffer B and the entire sample (4 mL) was injected
onto the column. The column was washed with 5 CV of Buffer B and
the product eluted using a gradient using Buffer A. A gradient of
100-60% Buffer B over 2 CV, hold for 1 CV, 60-35% Buffer B over 13
CV, hold for 1 CV and then 0% Buffer B for 5 CV. The chromatography
flow rate was 2.0 mL/min (150 cm/hr) and the column elution
monitored by the absorbance at 280 nm. The chromatography elution
profiles were compared and selected fraction were analyzed by SDS
PAGE. Analysis of the pooled fractions corresponding to the main
product peak is summarized in Table 4, below:
TABLE-US-00005 TABLE 4 Effect of Sodium Sulfate Concentration on
Separation and Recovery of EPO-PEGs using Phenyl 650S..sup.1 Sodium
Step EPO- EPO- MBP.sup.6 Sulfate Yield.sup.3 PEG.sub.3.sup.4
PEG.sub.2.sup.5 [mcg/mg Aggregate.sup.7 Concentration.sup.2 [%] [%]
[%] EPO] [%] 0.5 M 61.5 96.6 3.4 <0.083 0 0.6 M 64.0 95.4 4.6
<0.04 0.22 0.7 M 64.2 95.4 4.6 <0.04 0.34 .sup.1EPO-PEG
(85.1% EPO-PEG.sub.3 and 14.9% EPO-PEG.sub.2 (Table 7). .sup.2The
sodium sulfate concentration of Buffer B. .sup.3The step yield was
calculated as the ratio of EPO-PEG protein recovered after HIC
chromatography (combined fractions) versus the amount of EPO-PEG
injected onto the column. .sup.4The percent of EPO-(SA-PEG-10
kDa).sub.3 in the main product peak. .sup.5The percent of
EPO-(SA-PEG-10 kDa).sub.2 in the main product peak. .sup.6The
amount of MBP protein in the main product peak as determine by the
ELISA versus the amount of EPO-PEG protein. .sup.7The amount of
aggregate EPO-PEG in the product peak observed by SEC.
[0318] Standard purification conditions (Buffer A: 25 mM sodium
phosphate, pH 7.5. Buffer B: 25 mM sodium phosphate, 0.6M sodium
sulfate, pH 7.5. With 0.05 mg EPO-(SA-PEG-10 kDa).sub.1-4/mL resin
loaded) were compared to purification runs with buffer B prepared
with 0.5 and 0.7 M sodium sodium sulfate. Elution profiles (A280)
were compared. The run with buffer B containing 0.5 M Na sulfate
elutes earliest, followed by the 0.6 M Na sulfate and finally 0.7 M
Na sulfate run. The X-axis of these A280 traces were shifted to
overlay the peaks. Comparison of the X-axis shifted peaks shows
similar peak traces with respect to the elution of EPO-(SA-PEG-10
kDa).sub.2, EPO-(SA-PEG-10 kDa).sub.3, and EPO-(SA-PEG-10
kDa).sub.4. A slight difference in peak width was noticed when
comparing the elution peak profiles. Buffer B containing 0.7 M Na
sulfate shows the sharpest peak shape, followed by the 0.6 M Na
sulfate run and then 0.5 M Na sulfate run. The gradient programmed
on the chromatography system was kept constant for each run.
However, the increase or decrease of sodium sulfate in buffer B
will decrease or increase the actual slope of the elution gradient
respectively.
[0319] Table 4 compares EPO-(SA-PEG-10 kDa).sub.3 recovery,
MBP-ELISA, RP-HPLC, and SEC analysis data. No significant
difference was noted between the EPO-(SA-PEG-10 kDa).sub.3 purified
within the 0.5 to 0.7 M sodium sulfate concentration range.
Recovery is between 61.5 and 64.2%. ELISA indicates <0.04-0.083
mcg MBP/mg EPO-(SA-PEG-10 kDa).sub.3 remaining RP-HPLC analysis
showed that the amount of EPO-(SA-PEG-10 kDa).sub.2 remaining in
the EPO-(SA-PEG-10 kDa).sub.3 peak is between 3.4 and 4.6%.
Integration of the EPO-(SA-PEG-10 kDa).sub.4 peak was difficult at
low levels but the glycoform was detected by silver stain SDS-PAGE.
SDS-PAGE analysis showed that the purity of each EPO-(SA-PEG-10
kDa).sub.3 pool is similar as determined by colloidal blue as well
as silver stained gels.
Column Capacity (Phenyl 650S) for EPO-PEG.
[0320] The resin capacity for EPO-PEG and the ability of the Phenyl
650S chromatography process to separate the PEG states of EPO-PEG
was examined. A Tricorn 10 column was packed with 15.7 mL (1.0 cm
id.times.20 cm) of Phenyl 650S. The column was equilibrated with 5
column volumes (CV) of buffer B (25 mM sodium phosphate, 0.6 M
sodium sulfate, pH 7.5). In separate experiments, the amount of
EPO-PEG used in the process was varied (0.05, 0.1, 0.2, 0.5 mg
EPO-(SA-PEG-10 kDa).sub.1-4 protein/mL resin). The EPO-PEG.sub.x
mixture was adjusted to a sodium sulfate concentration of 0.6 M and
a final volume of 4 mL. This solution was injected onto the column.
The column was washed with 5 CV of Buffer B and the product eluted
using a gradient using Buffer A. A gradient of 100-60% Buffer B
over 2 CV, hold for 1 CV, 60-35% Buffer B over 13 CV, hold for 1 CV
and then 0% Buffer B for 5 CV. The chromatography flow rate was 2.0
mL/min (150 cm/hr) and the column elution monitored by the
absorbance at 280 nm. The chromatography elution profiles were
compared. Fractions were pooled and analyzed by SDS-PAGE. Analysis
of the main product peak is summarized in Table 5, below:
TABLE-US-00006 TABLE 5 Effect of Phenyl 650S Column Capacity on
Separation and Recovery of EPO-PEGs..sup.1 MBP.sup.6 Step EPO- EPO-
[mcg/ Injected Amount of Yield.sup.3 PEG.sub.3.sup.4
PEG.sub.2.sup.5 mg Aggregate.sup.7 EPO-PEG.sup.2 [%] [%] [%] EPO]
[%] 0.05 mg/mL resin 61.9 96.3 3.7 0.085 0.22 0.1 mg/mL resin 67.6
95.8 4.2 <0.024 0 0.2 mg/mL resin 70.2 94.9 5.1 <0.024 0 0.5
mg/mL resin 70.2 92.5 7.5 <0.019 0 .sup.1EPO-PEG (85.1%
EPO-PEG.sub.3 and 14.9% EPO-PEG.sub.2 (Table 7). .sup.2The ratio of
EPO-PEG protein injected per mL of HIC resin packed in the column
(mg EPO/mL resin). .sup.3The step yield was calculated as the ratio
of total EPO-PEG conjugate recovered after HIC chromatography
versus the amount of EPO-PEG injected onto the column. .sup.4The
percent of EPO-(SA-PEG-10 kDa).sub.3 in the main product peak.
.sup.5The percent of EPO-(SA-PEG-10 kDa).sub.2 in the main product
peak. .sup.6The amount of MBP-protein contained in the main product
peak as determine by ELISA assay versus the amount of EPO-PEG
protein. .sup.7The amount of aggregate EPO-PEG in the product peak
observed by SEC.
[0321] The robustness of the Phenyl 650S chromatography step was
tested with respect to variations in the quantity of EPO-(SA-PEG-10
kDa).sub.1-4 loaded per mL Phenyl 650S resin. Standard purification
conditions (Buffer A: 25 mM sodium phosphate, pH 7.5. Buffer B: 25
mM sodium phosphate, 0.6M sodium sulfate, pH 7.5 (0.05 mg
EPO-(SA-PEG-10 kDa).sub.1-4/mL resin loaded) were compared to
purification runs where the column was loaded with 0.1, 0.2 and 0.5
mg EPO-(SA-PEG-10 kDa).sub.1-4/mL resin. Comparison of the
resulting elution profiles (A280) shows a decrease in resolution
between the EPO-(SA-PEG-10 kDa).sub.2 and EPO-(SA-PEG-10 kDa).sub.3
peaks as the amount of protein loaded onto the column is increased.
RP-HPLC analysis was used to compare the amount of EPO-(SA-PEG-10
kDa).sub.2 remaining in the EPO-(SA-PEG-10 kDa).sub.3 pooled peak.
Table 5 compares EPO-(SA-PEG-10 kDa).sub.3 recovery, MBP-ELISA,
RP-HPLC, and SEC analysis data. No significant difference was noted
between the EPO-(SA-PEG-10 kDa).sub.3 purified within the
investigated column loading range. Recovery was between 61.9 and
70.2% (BCA analysis). As the quantity of material loaded onto the
column is increased the recovery increased. However, this increase
is also correlated with an increase in the amount of EPO-(SA-PEG-10
kDa).sub.2 remaining MBP-ELISA indicates <0.019-0.085 mcg MBP/mg
EPO-(SA-PEG-10 kDa).sub.3 remaining RP-HPLC analysis resulted in a
% EPO-(SA-PEG-10 kDa).sub.3 to % EPO-(SA-PEG-10 kDa).sub.2 ratio of
96.3:3.7, 95.8:4.2, 94.9:5.1, 92.5:7.5 for the 0.05 mg, 0.1 mg, 0.2
mg and 0.5 mg EPO-(SA-PEG-10 kDa).sub.1-4/mL resin loads,
respectively.
[0322] Increasing the quantity of the protein loaded onto the
column increases the amount of EPO-(SA-PEG-10 kDa).sub.2 in the
EPO-(SA-PEG-10 kDa).sub.3 peak since the peaks were pooled in the
same manner. Integration of the EPO-(SA-PEG-10 kDa).sub.4 was
difficult at low levels but the glycoform was detected by silver
stain SDS-PAGE. No aggregation of EPO-(SA-PEG-10 kDa).sub.3 was
detected by SEC analysis. Minor aggregation (0.22%) of
EPO-(SA-PEG-10 kDa).sub.3 was detected by SEC analysis in the 0.05
mg EPO-(SA-PEG-10 kDa).sub.1-4/mL resin load (this is the same
sample described in the sodium phosphate buffer concentration
experiment). SDS-PAGE analysis showed that the purity of each
EPO-(SA-PEG-10 kDa).sub.3 pool is similar when determined by
colloidal blue and silver stained gels.
Comparison of Phenyl 650S and 650M Resins
[0323] The ability of Phenyl 650M and Phenyl 650S chromatography
resins to separate the PEG states of EPO-PEG was examined. A
Tricorn 10 column was packed with 15.7 mL (1.0 cm id.times.20 cm)
of either Phenyl 650S or Phenyl 650M. Each column was equilibrated
with 5 column volumes (CV) of buffer B (25 mM sodium phosphate, 0.6
M sodium sulfate, pH 7.5). The EPO-PEG.sub.x (0.8 mg EPO protein; 1
mL) solution was adjusted to a sodium sulfate concentration of 0.6
M by the addition of 0.4 mL of Buffer A (25 mM sodium phosphate, pH
7.5) and 0.6 mL of buffer (2 M sodium sulfate, 25 mM sodium
phosphate, pH 7.5). This solution was then diluted with an equal
volume of Buffer B and the entire sample (4 mL) was injected onto
the column. The column was washed with 5 CV of Buffer B and the
product eluted using a gradient using Buffer A. A gradient of
100-60% Buffer B over 2 CV, hold for 1 CV, 60-35% Buffer B over 13
CV, hold for 1 CV and then 0% Buffer B for 5 CV. The chromatography
flow rate was 2.0 mL/min (150 cm/hr) and the column elution
monitored by the absorbance at 280 nm. The chromatography elution
profiles were compared and selected fractions were analyzed by SDS
PAGE. Analysis of the main product peak is summarized in Table 6,
below:
TABLE-US-00007 TABLE 6 Comparison of Phenyl 650S and 650M Resins
and the Effect on Separation and Recovery of EPO-PEGs..sup.1 Step
EPO- EPO- MBP.sup.6 Yield.sup.3 PEG.sub.3.sup.4 PEG.sub.2.sup.5
[mcg/mg Aggregate.sup.7 Resin.sup.2 [%] [%] [%] EPO] [%] Phenyl
650S 61.6 97.5 2.5 0.083 0 Phenyl 650M 65.1 93.6 6.4 0
.sup.1EPO-PEG (85.1% EPO-PEG.sub.3 and 14.9% EPO-PEG.sub.2 (Table
7). .sup.2Tosohaas Phenyl chromatography resins. .sup.3The step
yield was calculated as the ratio of EPO-PEG protein recovered
after HIC chromatography (combined fractions) versus the amount of
EPO-PEG injected onto the column. .sup.4The percent of
EPO-(SA-PEG-10 kDa).sub.3 in the main product peak. .sup.5The
percent of EPO-(SA-PEG-10 kDa).sub.2 in the main product peak.
.sup.6The amount of MBP protein contained in the main product peak
as determine by ELISA versus the amount of EPO-PEG protein.
.sup.7The amount of aggregate EPO-PEG in the product peak observed
by SEC.
[0324] The Phenyl 650S (35 micron bead size) chromatography step
was compared with Phenyl 650M (65 micron bead size) using standard
purification conditions (Buffer A: 25 mM sodium phosphate, pH 7.5.
Buffer B: 25 mM sodium phosphate, 0.6M sodium sulfate, pH 7.5 (0.05
mg EPO-(SA-PEG-10 kDa).sub.1-4/mL resin loaded). Comparison of the
resulting elution profiles showed that Phenyl 650S has a better
resolution between EPO-(SA-PEG-10 kDa).sub.2 and EPO-(SA-PEG-10
kDa).sub.3. Comparison of the respective EPO-(SA-PEG-10 kDa).sub.3
peak pools by SDS-PAGE showed similar purity except that the Phenyl
650S purified material appears to have less EPO-(SA-PEG-10
kDa).sub.4. Analysis by RP-HPLC showed a EPO-(SA-PEG-10 kDa).sub.3
to EPO-(SA-PEG-10 kDa).sub.2 ratio of 96.3:3.7 for Phenyl 650S and
a ratio of 93.6:6.4 for the Phenyl 650M purification.
[0325] The total system and column pressure measurements show the
average pressure throughout the Phenyl 650M purification was
approximately 0.12 mPa, while the Phenyl 650S pressure was
approximately 0.29 mPa.
Phenyl 650S vs Phenyl Sepharose HP Chromatography.
[0326] EPO-(SA-PEG-10 kDa).sub.3 purification using Phenyl
Sepharose HP (34 micron beads size) was performed using standard
purification conditions (Buffer A: 25 mM sodium phosphate, pH 7.5.
Buffer B: 25 mM sodium phosphate, 0.6M sodium sulfate, pH 7.5, with
0.05 mg EPO-(SA-PEG-10 kDa).sub.1-4/mL resin loaded. The elution
profile showed no actual resolution between EPO-(SA-PEG-10
kDa).sub.i, EPO-(SA-PEG-10 kDa).sub.2, EPO-(SA-PEG-10 kDa).sub.3
and EPO-(SA-PEG-10 kDa).sub.4 (chromatogram not shown). Since there
was no resolution between the various PEG states no analysis was
performed on the eluted material.
Example 2
Large-Scale Purification of GlycoPEGylated EPO using Hydrophobic
Interaction Chromatography
[0327] In order to further purify glycoPEGylated EPO from a
glycoPEGylation reaction mixture, and to separate the
trifunctionalized EPO species [EPO-(SA-PEG-10 kDa).sub.3] from
other PEGylated species (e.g., mono-, di- and tetra-PEGylated EPO
species), the mixture (e.g., flowthrough from an anion
chromatography medium) is subjected to hydrophobic interaction
chromatography (HIC). An exemplary HIC procedure is outlined below:
An XK26 column was packed with Phenyl 650S resin (106 mL, 2.6
cm.times.20 cm) and attached to an AKTA Explorer 100 system.
Product elution was monitored by absorbance at 214, 254 and 280 nm.
The column was equilibrated with 5 column volumes (CV) of Buffer B
(25 mM sodium phosphate, 0.6 M sodium sulfate, pH 7.5).
[0328] The solution of Sartobind Q purified EPO-(SA-PEG-10
kDa).sub.1-4 (62 mL, pH 6.94, 2.59 mS/cm) was adjusted to 0.6 M
sodium sulfate by dilution with 62 mL of 1.2 M of buffer (1.2 M
sodium sulfate, 25 mM sodium phosphate, pH 7.5). This solution (124
mL) was injected on the column. The ratio of EPO-PEG to resin upon
injection onto the column was 0.18 mg EPO protein per mL of resin
in the column. Any unbound material was washed from the column
using 5 CV of Buffer B (25 mM sodium phosphate, 0.6 M sodium
sulfate, pH 7.5). The EPO-(SA-PEG-10 kDa).sub.3 was eluted from the
column using the following gradient elution procedure using Buffer
A (25 mM sodium phosphate, pH 7.5). Gradient: 100-60% Buffer B over
2 CV, hold for 1 CV, 60-35% Buffer B over 13 CV (gradient change
1.92% Buffer B/CV), hold 1 CV and then 0% Buffer B for 5 CV. The
equilibration, load and wash steps were performed at a flow rate of
8 mL/min (90 cm/hr).
[0329] Flow through and wash fractions were collected in a 1 L
Nalgene container and individual elution fractions (12 mL) were
collected in 14 mL Falcon tubes. The elution profile and fraction
indicators are shown in FIGS. 2A and 2B. Fractions were combined,
the buffer was exchanged and the pool was concentrated using
Pellicon-2 XL 50 cm.sup.2. The pooled fractions containing the
EPO-(SA-PEG-10 kDa).sub.3 (pool F) were analyzed for protein
content (BCA, UV), identity (SDS-PAGE), glycosyltransferases
(MBP-ELISA), purity and EPO-PEG forms (RP-HPLC), aggregation and
purity (SEC) and endotoxin. Results are summarized in Table 7,
below:
TABLE-US-00008 TABLE 7 Results from the Large Scale Purification of
EPO-PEG using Phenyl 650S Chromatography Step EPO- EPO- MBP.sup.6
Amount Yield.sup.3 PEG.sub.3.sup.4 PEG.sub.2.sup.5 [mcg/mg Process
Step Injected.sup.2 [%[ [%[ [%[ EPO] Sartobind Q FT.sup.8 NA.sup.9
94.2 86.6 13.4 0.69 EPO-PEG.sub.3 18 mg 79.3 96.4 3.6 <0.0196
Phenyl 650S Fraction.sup.10 .sup.2Amount of EPO protein determined
by A280. .sup.3The step yield was calculated as the ratio of
EPO-PEG protein recovered after HIC chromatography (combined
fractions) versus the EPO-PEG injected onto the column. .sup.4The
percent of EPO-(SA-PEG-10 kDa).sub.3 in the main product peak.
.sup.5The percent of EPO-(SA-PEG-10 kDa).sub.2 in the main product
peak. .sup.6The amount of MBP protein contained in the main product
peak as determine by ELISA versus the amount of EPO-PEG protein.
.sup.7The amount of aggregate EPO-PEG in the product peak observed
by SEC. .sup.8EPO-PEG filtered through a Sartobind Q resin.
.sup.9Not available. .sup.10Combined fractions of the main product
peak after chromatography using optimized conditions on a Phenyl
650S resin.
[0330] Phenyl 650S Chromatography was scaled up from a 0.8 mg
EPO-(SA-PEG-10 kDa).sub.1-4 load (15.7 mL column) to a 22.4 mg load
(106 mL column). The standard conditions were used except the
amount of material loaded on the column was increased from 0.05 to
0.2 mg EPO-(SA-PEG-10 kDa).sub.1-4/mL resin. The EPO-(SA-PEG-10
kDa).sub.3 peak was pooled as shown in FIG. 2B resulting in a 408
mL peak pool (3.85 CV). Prior to concentration 16.1 mg
EPO-(SA-PEG-10 kDa).sub.3 was recovered (72%) with 5.7%
EPO-(SA-PEG-10 kDa).sub.2 remaining from the original 14.9%
EPO-(SA-PEG-10 kDa).sub.2 in the load material. Concentration and
diafiltration into 10 mM sodium acetate, 150 mM NaCl, pH 5.4 using
a Pellicon-2 XL 50 cm.sup.2 yielded a 95% recovery.
Example 3
Preparation and Isolation of EPO-(SA-PEG-10 kDa).sub.3 from a
GlycoPEGylation Reaction Mixture
[0331] This example summarizes the results obtained with a 3
chromatography step (and two ultrafiltration/diafilatration (UF/DF)
steps) purification process for the isolation of baculoviral
derived, glycoPEGylated human erythropoietin EPO-(SA-PEG-10
kDa).sub.3 (see, description of EPO-PEG conjugates in Example 1,
above) at a 20 mg scale. Overall process efficiency and product
quality were assessed.
[0332] The purification process (see e.g., FIG. 1) began with a
Sartobind Q membrane used in a negative binding mode which allowed
the PEG-EPO conjugates to flow through while capturing
glycoPEGylation enzymes, such as MBP-GnT1, MBP-GalT1,
MBP-SBD-ST3Gal3 and other enzyme contaminants. The various PEG
species generated in the glycoPEGylation reaction were then
fractionated using HIC on a Phenyl 650S resin (also compare Example
1), which enriched the EPO-(SA-PEG-10 kDa).sub.3 to a concentration
of >96% (step yield approximately 75%). Tangential flow
filtration (TFF, ultrafiltration/diafiltration) employing
regenerated cellulose (10 kDa MWCO) membranes, was used to
concentrate and reduce the conductivity of the HIC elution pool.
Cation exchange chromatography using Source 15S was then employed
to remove remaining host cell proteins and to further enrich the
EPO-(SA-PEG-10 kDa).sub.3 to a concentration of greater than 97%
(80% step yield). Finally, TFF with regenerated cellulose, 10 kDa
MWCO membranes was used to concentrate and change the buffer for
storage of the bulk product. 6.7 mg of purified EPO-PEG conjugate
was obtained, which corresponds to an overall yield of 56% for the
purification process (after correction for sampling and small-scale
side experiments). Process parameters are summarized in Table 8,
below:
TABLE-US-00009 TABLE 8 Summary of Process Steps and Analytical
Results Total Total Protein RP-HPLC mcg MBP/mg Process Step Protein
Load Recovery Step Recovery (% Tri/Di PEG) PEG-EPO PEGylation ~25
mg.sup.1 21.7 mg.sup.2 100% 83/17 138.58 Reaction Mixture Sartobind
Q 20.9 mg.sup.2 19.7 mg.sup.2 94% .sup. 86.6/13.4.sup.3 0.69 Phenyl
650S 15.25 mg.sup.2 11.1 mg.sup.4 .sup. 73%.sup.5 -- -- TFF 11.02
mg.sup.4 12.1 mg.sup.4 110% 96.4/3.6 <0.0196 Source 15S 10
mg.sup.4 7.93 mg.sup.3 79% 97.1/2.9 -- TFF 7.89 mg.sup.4 7.75
mg.sup.4 98% -- -- 0.2um syringe 6.95 mg.sup.4 6.69 mg.sup.4 96% --
<0.0196 Filtration.sup.6 Total Process 56% Recovery The overall
process pool was reduced by sampling and/or for side experiments at
each step. .sup.1Protein concentration was determined by BCA
relative to BSA standard prior to initial buffer exchange.
.sup.2Protein concentration determined by RP-HPLC.
.sup.3GlycoPEGylation reaction continued slightly after the
original RP-HPLC analysis while the sample was awaiting
purification of Sartoind Q (<1 hour) .sup.4Protein concentration
determined by A280 (extinction coefficient 1.24) .sup.5Actual
protein concentration is too low for accurate measurement by A280
(0.025 mg/mL). Two step recovery (Phenyl 650S + TFF) = 79% .sup.6A
small percentage (2.2%) of the final filtration volume was held up
in the sterile filter.
[0333] At each chromatography step the PEG-EPO product pool was
analyzed by SDS-PAGE, RP-HPLC for PEG-state and MBP ELISA to track
the removal of enzyme related contaminants. The resulting
EPO-(SA-PEG-10 kDa).sub.3 product was subjected to available drug
product release tests. The purity was found to be greater than 99%
by HPLC (combined glycoforms). The concentration of EPO-(SA-PEG-10
kDa).sub.3 in the final composition was 96.9%. The concentration of
EPO-(SA-PEG-10 kDa).sub.2 was 2.5% and the concentration of
EPO-(SA-PEG-10 kDa).sub.4 was 0.6%. Other EPO-PEG glycoforms were
not detected in the final composition. There was less than 1%
aggregate by SEC.
GlycoPEGylation Reaction
[0334] Human EPO intermediate protein (produced by Baculovirus
fermentation and purified) was stored frozen in 20 mM HEPES, pH 7.5
at -20.degree. C. at a concentration of 1.29 mg/mL as determined by
BCA assay. MBP-GnT1 was stored frozen in 50 mM Tris, pH 7.0, 138 mM
NaCl at -20.degree. C. The reported activity assay value of 0.5
U/mL was used. RP-HPLC analysis determined the protein
concentration to be 0.3 mg/mL. MBP-GalT1 was stored frozen in 20 mM
HEPES, pH 7.5, 200 mM NaCl at -20.degree. C. The activity was
reported to be 15 U/mL, with a protein concentration of 1.0 mg/mL
as determined by RP-HPLC. MBP-SBD-ST3Gal3 was stored frozen in 20
mM HEPES, pH 7.0 at -20.degree. C. The reported activity was 2.05
U/mL and the protein concentration was measured to be 1.06 mg/mL by
BCA assay. UDP-GlcNAc and UDP-Gal were prepared as 60 mg/mL stock
solutions in 100 mM HEPES, pH 7.0, 20 mM NaCl, 0.02% NaN.sub.3
immediately prior to use. CMP-SA-PEG-10 kDa was prepared as a 200
mg/mL stock solution in 100 mM HEPES, pH 7.0, 20 mM NaCl, 0.02%
NaN.sub.3 immediately prior to use.
Preparation and Analysis of 25 mg EPO-(SA-PEG-10 kDa).sub.3
Reaction
[0335] EPO intermediate protein (25 mg, 19.4 mL, 1.25 micromoles by
BCA assay) was concentrated to a volume of 1.0 mL in a Centricon
Plus-20 centrifugal filter (5 kDa MWCO) and then diluted with 15 mL
of 100 mM HEPES, pH 7.0, 20 mM NaCl, 0.02% NaN.sub.3. The EPO
solution was concentrated to 0.94 mL (26.7 mg/mL). MBP-SBD-ST3Gal3
(3.05 mL, 6.25 U) was diluted with 13 mL of 100 mM HEPES, pH 7.0,
20 mM NaCl, 0.02% NaN.sub.3 in another Centricon Plus-20
centrifugal filter (5 kDa MWCO) and was concentrated to 0.51 mL
(12.3 U/mL). The UDP-GlcNAc (25 micromoles, 0.27 mL) and UDP-Gal
(25 micromoles, 0.25 mL) stock solutions (both at 60 mg/mL in 100
mM HEPES, pH 7.0, 20 mM NaCl, 0.02% NaN.sub.3), MBP-GnT1 (0.5 mL,
0.25 U), MBP-GalT1 (0.33 mL, 5 U), the concentrated MBP-SBD-ST3Gal3
(0.51 mL of 12.3 U/mL, 6.25 U, from above), CMP-SA-PEG-10 kDa (25
micromole, 1.25 mL of 200 mg/mL solution in 100 mM HEPES, pH 7.0,
20 mM NaCl, 0.02% NaN.sub.3) and MnCl.sub.2 (5 mM, 0.104 mL of 200
mM solution in water) were combined. Additional 100 mM HEPES, pH
7.0, 20 mM NaCl, 0.02% NaN.sub.3 buffer was added (1.0 mL) to bring
the total volume to 4.17 mL. The reaction was incubated at
32.degree. C. for 2 hrs without shaking The conversion yield of
EPO-(SA-PEG-10 kDa).sub.3 was determined by SDS-PAGE and C3 RP-HPLC
(compare FIG. 4A). The reaction mixture was immediately purified on
a Sartobind Q cartridge as described below.
Sartobind Q Purification of PEG-EPO GlycoPEGylation Reaction
Mixture
[0336] A Sartobind Q SingleSep Nano (1 mL) cartridge was attached
to an AKTA
[0337] Explorer 100 system continuously monitoring absorbance at
214, 254 and 280 nm. The cartridge was flushed with 20 mM HEPES, 1
M NaCl, pH 7.0 (approximately 100 mL), followed by 20 mM HEPES, 20
mM NaCl, pH 7.0 (approximately 100 mL) both at a flow rate of 15
mL/min. The PEG-EPO reaction mixture (approximately 25 mg, 6 mg/mL,
4.2 mL) was diluted with 20 mM HEPES, 20 mM NaCl, pH 7.0 (22.8 mL)
to a final volume of 27 mL. A 1 mL aliquot was retained for
analysis and the concentration was determined to be 0.804 mg/mL by
RP-HPLC. The remaining diluted sample (26 mL, 20.9 mg by RP-HPLC)
was loaded on to the capsule and washed with 20 mM HEPES, 20 mM
NaCl, pH 7.0 (70 mL) at a flow rate of 15 mL/min.
[0338] Fractions were combined into a PEG-EPO product pool (80 mL).
Samples of the pools and selected individual fractions across the
flow through/wash product peak were analyzed by SDS-PAGE with
iodine and nd silver stain. The conjugate recovery for this step
(94%) was determined by RP-HPLC (Table 8).
[0339] The PEG-EPO pool was analyzed by MBP-ELISA for residual
enzyme contaminants (Table 8) and then divided into two portions:
The majority fraction (62 mL, 15.25 mg) was purified by HIC
chromatography on Phenyl 650S resin as described below. A small
sample (18 mL, 4.4 mg) was purified by an alternate process
including Fluoroapatite chromatography.
[0340] Bound impurities were eluted from the column with 20 mM
HEPES, 1 M NaCl, pH 7.0 (13 mL) at a flow rate of 15 mL/min.
Isolation of EPO-(SA-PEG-10 kDa).sub.3 from the Sartobind Q Flow
Through/Wash Pool using HIC (Phenyl 650S)
[0341] An XK26 column was packed with Phenyl 650S resin (106 mL,
2.6 cm.times.20 cm) as described herein above and was attached to
an AKTA Explorer 100 system continuously monitoring absorbance at
214, 254 and 280 nm. The column was equilibrated with 5 column
volumes (CV) 25 mM sodium phosphate, 0.6M sodium sulfate, pH 7.5
(212 mL). The Sartobind Q PEG-EPO product pool (62 mL, 18 mg, 0.17
mg PEG-EPO/mL resin, pH 6.94, 2.59 mS/cm) was diluted 1:1 with 1.2
M sodium sulfate, 25 mM sodium phosphate pH 7.5 (62 mL) to adjust
the sodium sulfate concentration to 0.6 M. The conditioned sample
(124 mL) was applied to the column. Unbound material was washed
from the column using 5 CV of 25 mM sodium phosphate, 0.6M sodium
sulfate, pH 7.5 (212 mL) at a flow rate of 8 mL/min (90 cm/hr). The
PEG-EPO species were fractionated and eluted with the following
gradient using Buffer A (25 mM sodium phosphate, pH 7.5) and Buffer
B (25 mM sodium phosphate, 0.6M sodium sulfate, pH 7.5) at 8 mL/min
(90 cm/hr): 100-60% B over 2 CV, isocratic hold at 60% B for 1 CV,
60-35% B over 13 CV (gradient change 1.92% B/CV), isocratic hold at
35% B for 1 CV, 35-0% B over 1 CV and 0% B for 5 CV. The flow
through and wash fraction were collected in the bottle from a 1 L
Nalgene filter unit and 12 mL elution fractions were collected in
14 mL Falcon tubes. Individual fractions were analyzed by SDS-PAGE.
Fractions enriched in EPO-(SA-PEG-10 kDa).sub.3 were combined
(after storage at 4.degree. C. for about 36 hours). Fractions from
other PEG-EPO species and elution peaks were also pooled. The
fraction pools were analyzed by SDS-PAGE. The dilute EPO-(SA-PEG-10
kDa).sub.3 Pool (444 mL) was analyzed by A280 (0.025 mg/mL, 11.1
mg) and the step recovery was determined to be 73% (Table 1). This
product pool was buffer exchanged and concentrated using Pellicon-2
XL regenerated cellulose membranes. The remaining 10 mg of material
(19.44 mL) was held on ice prior to further purification by cation
exchange chromatography on a Source 15S resin later the same day as
described below.
Purification of the Concentrated and Diafiltered Phenyl 650S
EPO-(SA-PEG-10 kDa).sub.3 Pool on Source 15S Chromatography
[0342] A Tricorn 10 column was packed with Source 15S (15.7 mL, 1
cm.times.20 cm) and attached to an AKTA Explorer 100 system
continuously monitoring absorbance at 214, 254 and 280 nm. The
column was equilibrated with 5 column volumes (CV) 10 mM Na
acetate, pH 5.4 (Buffer A). The concentrated and diafiltered Phenyl
650S EPO-(SA-PEG-10 kDa).sub.3 pool (10 mg PEG-EPO, 19.44 mL, pH
5.4, conductivity: 1.54 mS/cm) was divided into two equal portions
which were purified on the prepared column in two separate but
identical injections. Half of the diafiltered Phenyl 650S
EPO-(SA-PEG-10 kDa).sub.3 product pool (5 mg PEG-EPO, 9.72 mL each,
0.32 mg PEG-EPO/mL resin) was applied to the column at a flow rate
of 300 cm/h (1.96 mL/min). Unbound material was washed from the
column using 5 CV of 10 mM Na acetate, pH 5.4 (Buffer A). The
EPO-(SA-PEG-10 kDa).sub.3 was eluted with the following gradient
with Buffer B (0.5 M NaCl, 10 mM Na acetate, pH 5.4): 0-30% B over
24 CV, isocratic hold at 30% B for 1 CV, step to 100% B for 5 CV.
All steps were performed at 300 cm/h (1.96 mL/min). Fractions (1.6
mL) were collected in 96-deep well microtiter plates and stored at
4.degree. C. Fractions from both injections were pooled. Protein
content analysis (A280) of the EPO-(SA-PEG-10 kDa).sub.3 pool
indicated that the step recovery over the Source 15S chromatography
step was 79% (Table 8). RP-HPLC analysis of the product pool
indicated that the EPO-(SA-PEG-10 kDa).sub.2 impurity had been
reduced to 2.9% (Table 8). The product pool had an estimated sodium
chloride concentration of approximately 70 mM and was concentrated
and buffer exchanged. The Source 15S Flow Through/Wash pool was
electroblotted onto PVDF membranes and subjected to Edman
amino-terminal sequencing as described herein to determine the
identity of the major band in the pool. The amino-terminal sequence
obtained was determined to be EPO.
[0343] 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 in their entirety for all
purposes.
* * * * *