U.S. patent application number 11/483033 was filed with the patent office on 2006-11-09 for analysis and separation of platelet-derived growth factor proteins.
This patent application is currently assigned to Chiron Corporation. Invention is credited to Michael Kunitani, Hugh Parker, An D. Tran.
Application Number | 20060252695 11/483033 |
Document ID | / |
Family ID | 21866458 |
Filed Date | 2006-11-09 |
United States Patent
Application |
20060252695 |
Kind Code |
A1 |
Kunitani; Michael ; et
al. |
November 9, 2006 |
Analysis and separation of platelet-derived growth factor
proteins
Abstract
Methods for improving purification and quantification of
platelet derived growth factor (PDGF) proteins having structural
heterogeneity are provided. Preparation of substantially pure
isoforms of these proteins is achieved using TSK sulfopropyl cation
exchange chromatography and reverse phase high performance liquid
chromatography. A reverse charged capillary zone electrophoresis
method enables quantification of substantially pure isoforms of
these proteins resulting from endoproteolytic post-translational
modifications. Compositions of the invention are substantially
purified isoforms of secreted PDGF proteins having structural
heterogeneity, more particularly purified intact, single-clipped,
and double-clipped isoforms of recombinant PDGF-BB. Pharmaceutical
compositions comprising at least one of these substantially
purified recombinant PDGF isoforms and methods for their use in
promoting wound healing are also provided.
Inventors: |
Kunitani; Michael; (Corte
Madera, CA) ; Tran; An D.; (Moraga, CA) ;
Parker; Hugh; (Oakland, CA) |
Correspondence
Address: |
CHIRON CORPORATION
INTELLECTUAL PROPERTY - R440
PO BOX 8097
EMERYVILLE
CA
94662-8097
US
|
Assignee: |
Chiron Corporation
Emeryville
CA
|
Family ID: |
21866458 |
Appl. No.: |
11/483033 |
Filed: |
July 7, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10205693 |
Jul 26, 2002 |
7084262 |
|
|
11483033 |
Jul 7, 2006 |
|
|
|
09549290 |
Apr 14, 2000 |
6448382 |
|
|
10205693 |
Jul 26, 2002 |
|
|
|
08989250 |
Dec 12, 1997 |
6083910 |
|
|
09549290 |
Apr 14, 2000 |
|
|
|
60032720 |
Dec 13, 1996 |
|
|
|
Current U.S.
Class: |
514/8.2 ;
435/254.21; 435/69.1; 530/399 |
Current CPC
Class: |
A61K 9/0014 20130101;
A61P 19/08 20180101; C12N 15/81 20130101; C07K 14/49 20130101; A61L
2300/414 20130101; C07K 14/65 20130101; A61L 26/0066 20130101; C07K
2319/02 20130101; A61K 38/1858 20130101; A61P 17/02 20180101; A61K
9/06 20130101 |
Class at
Publication: |
514/012 ;
530/399; 435/069.1; 435/254.21 |
International
Class: |
A61K 38/18 20060101
A61K038/18; C12P 21/06 20060101 C12P021/06; C12N 1/18 20060101
C12N001/18; C07K 14/49 20060101 C07K014/49 |
Claims
1. A method for preparing a composition enriched in at least one
desired isoform of biologically active recombinant PDGF (rPDGF)
dimeric protein or variant thereof from a mixture of isoforms of
said rPDGF dimeric protein or variant thereof, said method
comprising taking said mixture and enriching said mixture in said
desired isoform by reducing amounts of undesired isoforms in said
mixture to obtain said composition.
2. The method of claim 1, wherein said enriching is accomplished
using ion exchange chromatography, reverse phase high-pressure
liquid chromatography, or reverse charge capillary zone
electrophoresis.
3. The method of claim 2, wherein said rPDGF dimeric protein is
rPDGF-BB, wherein the rPDGF-B polypeptides in said rPDGF-BB are
native-sequence or variant rPDGF-B polypeptides that exhibit at
least about 80% sequence identity to native-sequence rPDGF-B
polypeptides.
4. The method of claim 3, wherein the rPDGF-B polypeptides are
native-sequence polypeptides.
5. The method of claim 4, wherein the rPDGF-B polypeptides have the
amino acid sequences of human rPDGF-B polypeptides.
6. The method of claim 3, wherein said rPDGF-BB is produced in a
yeast host cell.
7. The method of claim 6, wherein said yeast host cell is a
Saccharomyces yeast host cell.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 10/205,693, filed Jul. 26, 2002, which is a
continuation of U.S. application Ser. No. 09/549,290, filed Apr.
14, 2000, which is a divisional of U.S. application Ser. No.
08/989,250, filed Dec. 12, 1997, now U.S. Pat. No. 6,083,910, which
claims the benefit of U.S. application Ser. No. 60/032,720, filed
Dec. 13, 1996, the contents of which are herein incorporated by
reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of protein
analysis and separation, particularly to preparations of purified
isoforms of heterologous proteins having structural
heterogeneity.
BACKGROUND
[0003] Platelet-derived growth factor (PDGF), the primary mitogen
in serum for mesenchymal-derived cells, is stored in platelet
alpha-granules. Injury to blood vessels activates the release of
PDGF from these granules in the vicinity of the injured vessels.
This mitogen acts as a potent chemoattractant for fibroblasts and
smooth muscle cells, as well as monocytes and neutrophils. The
mitogenic activity of the localized PDGF results in proliferation
of these cells at the site of injury, contributing to the process
of wound repair.
[0004] Purified native platelet-derived growth factor (PDGF), a
glycoprotein of about 30,000 daltons, is composed of two
disulfide-linked polypeptide chains. Two forms of these chains,
designated A and B, have been identified. The native protein occurs
as the homodimer AA or BB or the heterodimer AB, or a mixture
thereof. A partial amino acid sequence for the PDGF-A chain has
been identified (Johnsson et al. (1984) EMBO J. 3:921-928) and
cDNAs encoding two forms of PDGF A-chain precursors have been
described (U.S. Pat. No. 5,219,759). The mature A-chain consists of
a 104 amino acid polypeptide that is derived by proteolytic
processing of a 211 amino acid precursor polypeptide. The cDNA
encoding the PDGF-B chain has also been described (Nature (1985)
316:748-750). The mature B-chain consists of a 109 amino acid
polypeptide that is derived by proteolytic processing of a 241
amino acid precursor polypeptide. The mature A and B chains of PDGF
show sequence identity of 51%, with the eight cysteine residues
being conserved in each of the chains (Johnsson et al. (1984) EMBO
J. 3:921-928).
[0005] In addition to proteolytic processing of the precursor
polypeptides into mature PDGF-A and PDGF-B chains, recombinant PDGF
(rPDGF) produced in a yeast host undergoes further
post-translational processing that results in a secreted mature
protein having considerable structural heterogeneity. This is true
for the heterodimer rPDGF-AB, and more particularly for the
homodimer rPDGF-BB.
[0006] When expressed in a yeast host for production of a bulk drug
substance, PDGF undergoes endoproteolytic cleavage, or so-called
clipping, of the B-chain between the Arg-32 and Thr-33 residues,
resulting in a bulk drug substance having a mixture of unclipped or
so-called intact rPDGF and clipped rPDGF that has been cleaved in
the B-chain between the Arg-32 and Thr-33 residues. In the case of
rPDGF-BB, one or both of the B-chains may be clipped, resulting in
single-clipped or double-clipped rPDGF-BB, respectively. Other
post-translational modifications of interest to the present
invention include exoproteolytic removal of C-terminal amino acids,
or so-called truncation, which may remove Arg-32 from clipped
B-chains and/or Thr-109 from intact and clipped B-chains. These
post-translational modifications lead to a number of structural
forms, or so-called isoforms, of rPDGF-BB present in the secreted
product. Methods of the present invention are directed to
separation and purification of the intact, single-clipped, and
double-clipped isoforms of rPDGF, more particularly rPDGF-BB.
[0007] The three dimeric forms of PDGF exhibit different binding
affinities for the two known PDGF receptor gene products, a and P.
The beta receptor recognizes PDGF B chain and is dimerized in the
presence of PDGF-BB. The alpha receptor recognizes PDGF B and A
chains and can be dimerized by PDGF-BB, PDGF-AA, and PDGF-AB (see,
for example, Abboud et al (1994) J. Cell. Phys. 158:140-150). The
amino acid residue region of PDGF-BB involved in binding or
activation of the receptor has been narrowed down to residues
Ile25-Phe37 (Giese et al. (1990) Mol. Cell. Biol. 10:5496-5501).
These residues include the Arg-32/Thr-33 site cleaved by
endoproteolytic processing during production of PDGF-BB by a yeast
host.
[0008] Proteinase-resistant mutants of recombinant PDGF-BB
exhibiting improved biological activity have been identified (Cook
et al. (1992) Biochem. J. 281:57-65; see also European Patent
Application No. 0 547 064 B1). These mutants prevent
endoproteolytic cleavage of the B-chains at Arg-32. Thus
elimination of endoproteolytic cleavage in this region apparently
leads to increased biological activity of the PDGF-BB produced in a
yeast host during the fermentation process. This increase in
biological activity is presumably associated with an increase in
the relative amount of unclipped, or so-called intact, PDGF-BB and
a decrease in the relative amount of clipped PDGF-BB in the
recombinant PDGF-BB product. Similarly, endoproteolytic cleavage in
this region of either or both of the B-chains within a rPDGF-BB
protein would be expected to yield a bulk drug substance having
different biological activity than found for a bulk drug substance
consisting solely of the intact rPDGF-BB.
[0009] Prior to the present invention, methods commonly used in the
art to purify recombinantly produced PDGF have not distinguished
between the various isoforms of PDGF resulting from
post-translational processing, including endoproteolytic cleavage
of the PDGF-B chain between Arg-32 and Thr-33. Prior art teaches
that structural isoforms resulting from post-translational
endoproteolytic cleavage could have varying degrees of biological
activity, and hence can effect the overall biological activity of
bulk drug substance produced by fermentation.
[0010] Methods are needed to separate and quantify the structural
isoforms of recombinantly produced PDGF such that their biological
activities can be compared. These methods would be useful in
preparing bulk drug substances consisting of pure isoforms.
SUMMARY OF THE INVENTION
[0011] Methods for improving purification and quantification of
platelet derived growth factor (PDGF) proteins having structural
heterogeneity are provided. Preparation of substantially pure
isoforms of these proteins is achieved using TSK sulfopropyl cation
exchange chromatography and reverse phase high performance liquid
chromatography. These methods are particularly useful for
separating isoforms that result from post-translational
endoproteolytic processing of secreted recombinant PDGF (rPDGF),
more particularly rPDGF-BB. A reverse charge capillary zone
electrophoresis method of the invention is useful for quantifying
endoproteolytic post-translational modifications.
[0012] Compositions of the invention are substantially purified
isoforms of secreted PDGF proteins having structural heterogeneity,
more particularly purified intact, single-clipped, and
double-clipped isoforms of PDGF-BB. Pharmaceutical compositions
comprising at least one of these substantially purified PDGF
isoforms and methods for their use in promoting wound healing are
also provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a schematic representation of intact isoforms of
rhPDGF-BB (group A) with or without truncation of Thr-109. Single
lines represent B chains; paired lines represent rhPDGF-BB.
[0014] FIG. 2 is a schematic representation of single-clipped
isoforms of rhPDGF-BB with or without truncation of Arg-32 and
Thr-109. Paired lines represent single-clipped rhPDGF-BB. The clip
in one of the B chains is represented by a break in the line.
Isoforms of single-clipped species are separated into groups B and
C based on the difference in the overall charge of the protein.
Truncation of Arg-32 at the clip site (group C) produces a more
acidic isoform, while truncation only of Thr-109 (group B) does not
alter the molecular charge.
[0015] FIG. 3 is a schematic representation of double-clipped
isoforms of rhPDGF-BB with or without truncation of Arg-32 and
Thr-109. Paired broken lines represent double-clipped rhPDGF-BB.
The clip in each of the B chains is represented by a break in each
line. The double-clipped isoform is separated into groups D, E, and
F based on the difference in the overall charge of the protein.
Truncation of the Arg-32 at one of the clip sites (group E) makes
the isoform more acidic, while truncation of Arg-32 at both of the
clip sites (group F) removes two basic amino acid residues.
Truncation only of Thr-109 (group D) does not alter the molecular
charge, similar to single-clipped species (group B).
[0016] FIG. 4 is a high temperature reverse phase (RP) HPLC profile
of rhPDGF-BB using a linear gradient elution. Double-clipped and
truncated double-clipped isoforms (A), single-clipped and truncated
single-clipped isoforms (B), and intact and truncated intact
isoforms (C) of rhPDGF-BB are resolved as three groups of several
peaks.
[0017] FIG. 5 is a high temperature RP HPLC profile of rhPDGF-BB
using a step gradient elution. Double-clipped and truncated
double-clipped isoforms (A), single-clipped and truncated
single-clipped isoforms (B), intact and truncated intact isoforms
(C), and a dimer/dimer (D) of rhPDGF-BB are resolved as individual
peaks.
[0018] FIG. 6 is an electropherogram of reverse charge capillary
zone electrophoresis (RC CZE) of carboxypeptidase B treated PDGF-BB
(6B) in comparison to non-treated rhPDGF-BB (6A). Letters
correspond to the groups identified in FIGS. 1-3.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention is directed to compositions having
PDGF activity that are useful as therapeutic agents in the
treatment of wounds. More specifically, these compositions comprise
at least one biologically active, substantially purified isoform of
a recombinant PDGF (rPDGF) protein. By "recombinant" is intended
the protein is produced as a heterologous or foreign protein in the
cells of a host organism stably transformed with a nucleotide
sequence encoding the foreign PDGF protein of interest.
[0020] Methods of the invention provide for the separation of
biologically active isoforms of rPDGF from a bulk drug substance
comprising a mixture of these isoforms. The methods are
particularly useful for separating isoforms that result from
post-translational endoproteolytic processing of secreted rPDGF,
more particularly rPDGF-BB. Once separated, isoforms can be
substantially purified and incorporated into a pharmaceutical
composition for use in treatment of wounds.
[0021] An isoform of a rPDGF protein is "biologically active" when
it has the capability of performing one or more biological
functions or a set of activities normally attributed to PDGF in a
biological context. These biological activities include inducing
chemotaxis and/or mitogenesis of responsive cell types following
the binding of PDGF to a specific cell surface receptor. Other
biological activities of PDGF may include, but are not limited to
phospholipase activation, increased phosphotidylinositol turnover
and prostaglandin metabolism, stimulation of both collagen and
collagenase synthesis by responsive cells, indirect proliferative
response of cells lacking PDGF receptors, and potent
vasoconstrictor activity. Biological activity can be determined
using any number of assays available in the art for PDGF, including
but not limited to the mitogenic assay described herein.
[0022] By "substantially purified isoform" is intended at least
about 80%, preferably at least about 90%, more preferably at least
about 95% of the composition comprising rPDGF is the isoform of
interest. Substantially purified isoforms of a protein are obtained
using methods of the present invention that provide for separation
of the isoform of interest from a cell culture comprising a mixture
of rPDGF isoforms. By "separation of the isoform of interest" is
intended removal of any undesirable isoforms or other components in
the cell culture such that the remaining isoform is substantially
purified.
[0023] The rPDGF protein whose isoforms are separated using methods
of the present invention will be a biologically active dimeric
form, more particularly the homodimer rPDGF-AA and PDGF-BB or the
heterodimer rPDGF-AB, as well as any substantially homologous and
functionally equivalent variants thereof. By "variant" is intended
a protein derived from the native protein by deletion or addition
of one or more amino acids to the N-terminal or C-terminal end of
the native protein; deletion or addition of one or more amino acids
at one or more sites in the native protein; or substitution of one
or more amino acids at one or more sites in the native protein.
Conservative substitutions that preserve the general charge,
hydrophobicity/ hydrophilicity, and/or steric bulk of the amino
acid substituted may be preferred. Such substitutions may be made,
for example, between the members of the following groups: GlyAla,
ValIleLeu, AspGlu, LysArg, AsnGln, and PheTrpTyr. Other
substitutions may be made to eliminate nonessential amino acid
residues, such as to alter a glycosylation site, a phosphorylation
site, an acetylation site, or to alter the folding pattern by
altering the position of a cysteine residue that is not necessary
for function. Methods for such manipulations are generally known in
the art.
[0024] For example, amino acid sequence variants of the PDGF
protein of interest can be prepared by mutations in the DNA
sequence encoding the native PDGF protein. Methods for mutagenesis
and nucleotide sequence alterations are well known in the art. See,
for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492;
Kunkel et al. (1987) Methods Enzymol. 154:367-382; U.S. Pat. No.
4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular
Biology (MacMillan Publishing Company, New York) and the references
cited therein; herein incorporated by reference. Such variants will
continue to possess the desired biological activity. Obviously, the
mutations that will be made in the DNA encoding the variant PDGF
protein must not place the sequence out of reading frame and
preferably will not create complementary regions that could produce
secondary mRNA structure. See EP Patent Application Publication No.
75,444.
[0025] Thus rPDGF proteins whose isoforms are separated using
methods of the present invention include the naturally occurring
PDGF proteins as well as variants thereof. These variants will be
substantially homologous and functionally equivalent to their
native PDGF protein. A variant of a native PDGF protein is
"substantially homologous" to the native protein when at least
about 80%, more preferably at least about 90%, and most preferably
at least about 95% of its amino acid sequence is identical to the
amino acid sequence of the native PDGF protein. Variants may differ
by about 1, 2, 3, or 4 amino acids. By "functionally equivalent" is
intended that the sequence of the variant defines a chain that
produces a protein having substantially the same biological
activity as the native protein of interest. Such functionally
equivalent variants that comprise substantial sequence variations
are also encompassed by the invention. Thus a functionally
equivalent variant of the native protein will have a sufficient
biological activity to be therapeutically useful. by
"therapeutically useful" is intended effective in achieving a
therapeutic goal, as, for example, healing a wound.
[0026] Methods are available in the art for determining functional
equivalence. Biological activity can be measured using assays
specifically designed for measuring activity of the native protein.
Additionally, antibodies raised against the biologically active
native protein can be tested for their ability to bind to the
functionally equivalent analog, where effective binding is
indicative of a protein having a conformation similar to that of
the native protein.
[0027] Methods of the present invention provide for improved
separation and purification of isoforms of rPDGF proteins, more
particularly those isoforms resulting from post-translational
endoproteolytic processing of rPDGF. The rPDGF whose isoforms are
separated may be the homodimer PDGF-BB or the heterodimer PDGF-AB.
In one preferred embodiment, the isoforms of recombinant human PDGF
(rhPDGF) protein are separated and purified using methods of the
invention.
[0028] The nucleotide sequence encoding the rPDGF protein whose
isoforms are separated using methods of the present invention may
be a genomic, cDNA, or synthetic DNA sequence. The genes encoding
the native forms of PDGF have been sequenced, and various analogs
are well known in the art. Expression of PDGF homodimers and
heterodimers is described in, for example, U.S. Pat. Nos.
4,766,073; 4,769,328; 4,801,542; 4,845,075; 4,849,407; 5,045,633;
5,128,321; and 5,187,263; herein incorporated by reference. Based
on the known amino acid sequences for the A- and B-chain
polypeptides, synthetic nucleotide sequences encoding PDGF A-chain
and B-chain polypeptides may be made in vitro using methods
available in the art. See particularly Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y.).
Where the recombinant PDGF protein of interest is the heterodimer
rhPDGF-AB, the nucleotide sequences encoding the hybrid precursor
polypeptides comprising the A- and B-chain polypeptides may be
assembled as part of one expression cassette or assembled into
separate expression cassettes for cotransformation of a host
cell.
[0029] The nucleotide sequence encoding a recombinant PDGF protein
of interest can be expressed in a suitable expression system for
subsequent separation and purification of isoforms of interest
according to methods of the present invention. Methods are
available in the art for expressing heterologous proteins, more
particularly for expressing recombinant PDGF homodimers and
heterodimers in bacteria (see, for example, Hoppe et al. (1990)
Eur. J. Biochem 187:207-214; Fretto et al. (1993) J. Biol. Chem.
268:3625-3631), yeast (Kelly et al. (1984) EMBO J. 4:3399-3405;
Ostman et al. (1989) Growth Factors 1:271-281), and mammalian cells
(see, for example, Ostman et al. (1988) J. Biol. Chem.
263:16202-16208). See also EP 177,957; U.S. Pat. Nos. 5,219,759 and
6,017,731; and the copending patent application entitled "Method
for Expression of Heterologous Proteins in Yeast," Ser. No.
09/340,250; herein incorporated by reference. Methods of the
present invention may be practiced with rPDGF produced in any host
system where post-translational endoproteolytic processing leads to
a secreted rPDGF product comprising a mixture of isoforms.
[0030] Problems with post-translational proteolytic processing and
generation of isoforms are frequently encountered in rPDGF produced
in a yeast host. By "yeast" is intended ascosporogenous yeasts
(Endomycetales), basidiosporogenous yeasts, and yeast belonging to
the Fungi Imperfecti (Blastomycetes). The ascosporogenous yeasts
are divided into two families, Spermophthoraceae and
Saccharomycetaceae. The later is comprised of four subfamilies,
Schizosaccharomycoideae (e.g., genus Schizosaccharomyces),
Nadsonioideae, Lipomycoideae, and Saccharomycoideae (e.g., genera
Pichia, Kluyveromyces, and Saccharomyces). The basidiosporogenous
yeasts include the genera Leucosporidium, Rhodosporidium,
Sporidiobolus, Filobasidium, and Filobasidiella. Yeast belonging to
the Fungi Imperfecti are divided into two families,
Sporobolomycetaceae (e.g., genera Sporobolomyces, Bullera) and
Cryptococcaceae (e.g., genus Candida). Of particular interest to
the present invention are species within the genera Pichia,
Kluyveromyces, Saccharomyces, Schizosaccharomyces, and Candida. Of
particular interest are the Saccharomyces species S. cerevisiae, S.
carlsbergensis, S. diastaticus, S. douglasii, S. kluyveri, S.
norbensis, and S. oviformis. Species of particular interest in the
genus Kluyveromyces include K. lactis. Since the classification of
yeast may change in the future, for the purposes of this invention,
yeast shall be defined as described in Skinner et al., eds. (1980)
Biology and Activities of Yeast (Soc. App. Bacteriol. Symp. Series
No. 9). In addition to the foregoing, those of ordinary skill in
the art are presumably familiar with the biology of yeast and the
manipulation of yeast genetics. See, for example, Bacila et al.,
eds. (1978) Biochemistry and Genetics of Yeast; Rose and Harrison,
eds. (1987) The Yeasts (2.sup.nd ed.); Strathern et al., eds.
(1981) The Molecular Biology of the Yeast Saccharomyces; herein
incorporated by reference.
[0031] The selection of suitable yeast and other microorganism
hosts for expression of heterologous proteins is within the skill
of the art. When selecting yeast hosts for expression, suitable
hosts may include those shown to have, inter alia, good secretion
capacity, low proteolytic activity, and overall robustness. Yeast
and other microorganisms are generally available from a variety of
sources, including the Yeast Genetic Stock Center, Department of
Biophysics and Medical Physics, University of California, Berkeley,
Calif.; and the American Type Culture Collection, Rockville,
Md.
[0032] Methods for expression of rPDGF in a yeast host are
available in the art. One such method comprises transforming a
yeast host cell with a plasmid expression vector comprising a
nucleotide sequence encoding human PDGF-BB. This method is briefly
described in Example 1 herein and is described in detail in U.S.
Pat. No. 6,017,731, and in the copending patent application
entitled "Method for Expression of Heterologous Proteins in Yeast,"
Ser. No. 09/340,250; herein incorporated by reference.
[0033] When expressed in a yeast host, rPDGF is secreted as a
complex mixture of structural isoforms that result from
post-translational processing of the protein during the
fermentation production process. Post-translational processing is
particularly prevalent in expression of rPDGF-BB. These structural
isoforms have varying degrees of biological activity, as
demonstrated using standard assays for PDGF activity. Methods of
the invention are directed to separation of these structural
isoforms such that a final pharmaceutical composition comprising
substantially purified isoforms of the greatest biological activity
can be prepared. These methods are useful for separating isoforms
of rPDGF-AB, and more particularly isoforms of rPDGF-BB.
[0034] Recombinant PDGF-BB produced in a yeast host cell is
secreted as a fully folded, biologically active homodimeric protein
consisting of two highly twisted antiparallel pairs of B chains
(Oefner et al. (1992) EMBO J. 11:3921-3926). Each B chain comprises
109 amino acid residues. During fermentation to produce a bulk drug
substance, several post-translational modifications occur to the
secreted rPDGF-BB. This results in a bulk drug substance comprising
rPDGF protein having considerable structural heterogeneity. Such
modifications include, but are not limited to endoproteolytic
digestion between residues Arg-32 and Thr-33 (referred to as
"clipping") and exoproteolytic removal of C-terminal amino acids
(referred to as "truncation") at Arg-32 and Thr-109, glycosylation
of Ser and/or Thr residues, and oxidation of methionine. These
post-translational modifications lead to a number of structural
forms, or so-called isoforms, of rPDGF-BB present in the secreted
product. The structural heterogeneity of the rPDGF-BB yeast product
is further complicated by the presence of a rigid Pro-Pro bond at
residues 41 and 42. The rotation of this rigid bond is further
hindered by its proximity to a disulfide bond and the bulky side
chain of Trp-40. This rotational hindrance leads to the formation
of stable cis-trans isomers of rPDGF-BB at room temperature.
Although numerous potential isoforms exist when considering the
multiple combinations of post-translational modifications and
Pro-Pro isomerization, isoforms resulting from post-translational
clipping and truncation are of primary interest to the present
invention.
[0035] Post-translational endoproteolytic processing or clipping
between Arg-32 and Thr-33 leads to three different and distinct
basic isoforms of rPDGF-BB: intact, single-clipped, and
double-clipped. By "intact" is intended both B chains remain intact
and are not endoproteolytically cleaved. By "single-clipped" is
intended one B chain is intact and one B chain is
endoproteolytically cleaved between Arg-32 and Thr-33. By
"double-clipped" is intended both B chains are endoproteolytically
cleaved between Arg-32 and Thr-33. Each respective isoform may be
additionally modified by C-terminal truncation of Arg-32 or
Thr-109, producing a known number of derived structural species for
any given basic isoform. Thus, for the intact isoform, C-terminal
truncation may occur only at residue 109, leading to three
different structural species of that isoform. For the
single-clipped isoform, C-terminal truncation of both residues 32
and 109 can produce eight different structural species of that
isoform. For the double-clipped isoform, ten structural species of
that isoform are possible. All together, the post-translational
processes of clipping followed by C-terminal truncation of Arg-32
or Thr-109 residues leads to a complex mixture of twenty-one
possible structural isoforms of rPDGF-BB in the bulk drug
substance, as illustrated schematically in FIGS. 1-3.
[0036] Methods of the invention are directed to separation of the
basic isoforms designated as intact, single-clipped, and
double-clipped isomers of rPDGF-BB, each of which comprises a known
number of derived structural species resulting from truncation of
the Arg-32 or Thr-109 residues. It has been determined that the
intact isoform has 100% mitogenic activity, the single-clipped
isoform has 120% mitogenic activity, and the double-clipped isoform
has 20% mitogenic activity. The reference sample exhibits 50%
activity. Accordingly, utilizing the methods of the invention
compositions can be obtained having at least about 20% greater,
preferably at least about 50% greater, more preferably at least
about 100% greater PDGF activity than the unpurified cell culture.
Thus, for purposes of preparing pharmaceutical rPDGF-BB
compositions having use as a therapeutic agent for treatment of
wounds, it is clearly desirable to separate these isoforms such
that the pharmaceutical compositions comprise intact and/or
single-clipped isoforms of rPDGF-BB. Methods of the invention
enable preparation of such pharmaceutical compositions by providing
for separation of the intact and single-clipped isoforms from the
double-clipped isoform of rPDGF-BB of the bulk drug substance
produced by fermentation.
[0037] Methods of the invention utilize ion exchange enrichment,
reverse phase high pressure liquid chromatography, and reverse
charge capillary zone electrophoresis. The first of these methods
is directed at separation of intact, single-clipped, and
double-clipped isomers of rPDGF, particularly rPDGF-BB, where
separation is to a degree of purification appropriate for
therapeutic use of the rPDGF. Hence, this method is appropriate for
separation of these isomers for use in production of a
pharmaceutical composition comprising substantially purified
intact, single-clipped, or intact and single-clipped rPDGF,
particularly rPDGF-BB. The second and third methods, while
providing a means for separation of the intact, single-clipped, and
double-clipped isomers, are primarily useful for analytical means,
as described below.
[0038] The intact and single-clipped isoforms of rPDGF-BB can be
separated by ion-exchange enrichment from the double-clipped
isoform to a degree of purification appropriate for therapeutic use
of rPDGF-BB. A bulk drug comprising biologically active rPDGF-BB
can be enriched in substantially purified intact and single-clipped
rPDGF-BB isoforms by separating these isoforms from the
double-clipped isoform using cation exchange chromatography and a
TSK sulfopropyl column (TSK SP). The bulk drug substance produced
by fermentation, which comprises all three of the isoforms, is run
on the column, and the desired isoforms are eluted with NaCl
solution. The double-clipped isoform elutes in the earlier
fractions, followed by the single-clipped and intact isoforms.
Removal of the eluted fraction comprising the double-clipped
isoform allows for subsequent collection of fractions eluting the
single-clipped and intact isoforms. These fractions can be used
separately, or together, to produce a composition comprising the
isolated intact isoform, single-clipped isoform, or a mixture of
the intact and single-clipped isoforms of rPDGF-BB.
[0039] Reverse phase high pressure liquid chromatography (RP HPLC)
is a method useful for separating certain isoforms of a dimeric
protein preparation having multiple isoforms, where at least one
isoform has two different faces. By "face" is intended a term used
in the context of RP HPLC, where each polypeptide chain of a
dimeric protein is a "face" of the molecule. For homodimers, like
PDGF-BB, where the two chains are identical and have no
modifications that make one face different than the other, the
molecule has but one face. However, where the molecule has, as in
the case of rPDGF-BB, a chain that is endoproteolytically cleaved
on one of the chains, thus making a single-clipped isoform, the
dimer resulting has two faces: the face of the unclipped chain and
the face of the clipped chain. A heterodimer naturally has two
faces. For example, in the case of the rPDGF-AB heterodimer, the A
chain is one face and the B chain is another.
[0040] For the purposes of the present invention, RP HPLC conducted
at high temperature is used to separate the intact, single-clipped,
and double-clipped isoforms of rPDGF-BB. In accordance with the
present invention, the RP HPLC method is conducted at a temperature
between about 75.degree. C. at a minimum and about 90.degree. C. at
a maximum, and more preferably the temperature is about 85.degree.
C., for separation of rPDGF-BB isoforms. The RP HPLC method of the
invention can further include using a combination isocratic/step
gradient of acetonitrile.
[0041] Prior to the present application of high temperature RP HPLC
to analyze and separate isoforms of rPDGF-BB, it had been noted by
researchers producing PDGF-BB in E. coli and analyzing it by RP
HPLC that at least 4 peaks were routinely observed in the resulting
chromatograms (see Watson and Kenney (1992) J. Chromatography
606:165-170). It was established that these peaks were due to
differences in exposed hydrophobicity caused by a Pro-Pro sequence
at residue 41 and 42 (Watson and Kenney (1992) J. Chromatography
606:165-170) and thus measured pro-pro conformers. Furthermore,
rPDGF-BB expressed in the yeast Saccharomyces cerevisae exhibits
multiple conformers that produce broad and heterogenous
chromatographic profiles when analyzed with RP HPLC at various
ambient and subambient temperatures, as disclosed in Example 3
below. Analysis and separation of rPDGF-BB by RP HPLC at these
temperatures is complicated by Pro-Pro isomerizations within the
molecule. Additionally, absorptive chromatography cannot
simultaneously resolve clipping and truncation to opposing faces of
the homodimeric protein under these conditions.
[0042] When conducted at high temperature in accordance with the
present invention, RP HPLC analysis accelerates the equilibrium of
Pro-Pro isomerization as well as accelerating the rate of
desorption. As a result, the phenomena of multiple Pro-Pro
conformers is not observed. Thus, the number of isoforms
potentially resolved can be reduced to the twenty-one structures
described earlier as intact, single-clipped, and double-clipped
isoforms and their exoproteolyzed species. Furthermore, when RP
HPLC analysis is carried out at about 85 C, the heterogeneity
resulting from the post-translational modifications due to clipping
and truncation occurring on both faces of the dimeric molecule can
be resolved.
[0043] A combination isocratic/step gradient can also be employed
in addition to the high temperature to improve separation of the
isoforms, thus greatly simplifying the chromatographic profile.
Using the high temperature RP HPLC method with the isocratic/step
gradient provides for separation of the isoforms of rPDGF-BB
predominantly on the basis of clipping, facilitating analysis and
separation of intact, single-clipped, and double-clipped isoforms
of rPDGF-BB. Methods for conducting an isocratic/step gradient are
available in the art.
[0044] Quantities of the intact, single-clipped, and double-clipped
isoforms of rPDGF-BB can be separated from a bulk drug substance
comprising a mixture of rPDGF isoforms using the RP HPLC method at
high temperature and an isocratic/step gradient of acetonitrile in
accordance with the present invention. These isoforms can be
purified from the acetonitrile and TFA solvent and buffer
components. Purification is achieved using standard means for
removing proteins from such solvents. For example, dialysis using
small tubes, or for larger amounts using continuous flow dialysis
fibers, can be conducted to yield a product suitable for use as a
therapeutic agent.
[0045] The purified rPDGF-BB isoforms obtained in accordance with
this method of the invention are useful for conducting biological
activity assays to determine relative biological activity of the
purified isoforms. In the case of rPDGF-BB, a mitogenic assay to
determine the biological activity of a bulk drug substance
comprising at least one isoform of rPDGF-BB can be conducted as
described in Example 5 disclosed herein.
[0046] While the high temperature RP HPLC method of the present
invention is useful for separating quantities of isoforms of
rPDGF-BB sufficient to analyze biological activity of individual
isoforms, the method is not suitable for routine analysis and
quantification of the intact and clipped isoforms in a bulk drug
substance. The third method of the invention, reverse charge
capillary zone electrophoresis (RC CZE) was developed to provide
for such quantification and routine analysis.
[0047] The reverse charge CZE of the present invention was
originally developed to monitor charge heterogeneity since the
extremely high isoelectric point (pI=10.5) of the rPDGF-BB molecule
precluded the use of conventional isoelectric focusing (IEF)
analysis. However, the use of hand cast very high pI IEF gels may
be possible.
[0048] Separation by RC CZE is based on charge/size ratio. Thus,
with respect to the rPDGF-BB of the present invention, truncation
of the basic residue Arg-32 has a greater effect on RC CZE assay
selectivity than truncation of the neutral amino acid residue
Thr-109. Since truncation of the Thr-109 residue does not
appreciably alter the charge or size of the protein, there is no
separation between the three possible categories of isoforms within
the intact isoform. The baseline separation observed between the
intact isoform and the single-clipped isoform may be due to an
increase in hydrodynamic size of the single-clipped isoform, which
may occur through partial denaturation of the secondary structure.
Within the single-clipped isoform, the separation of the two
species (one without the C-terminal truncation of Arg-32 and the
other with that truncation) is due to the decrease in charge of the
species of isoform resulting from C-terminal truncation of Arg-32.
Within the double-clipped isoforms, three isoforms can be
distinguished; the separation of them may be due to the charge
difference resulting from either a single or a double C-terminal
truncation of Arg-32.
[0049] The RC CZE method described thus far is unable to
distinguish between, and thus separate, single-clipped isoforms
that have the Arg-32 truncation and double-clipped isoforms that do
not have the Arg-32 truncation. In order to separate these two
isoform categories, and in order to quantify the intact,
single-clipped, and double-clipped isoforms, the RC CZE method of
the present invention further comprises treating the bulk drug
substance comprising the various rPDGF-BB isoforms with
carboxypeptidase B prior to analysis. This enzyme is specific for
the basic amino acids arginine and lysine located at the carboxy
terminus. Enzyme treatment converts single-clipped isoforms not
having an Arg-32 truncation to single-clipped isoforms having the
Arg-32 truncation, and converts double-clipped isoforms having no,
or only one, Arg-32 truncation to isoforms having two Arg-32
truncations. Thus, the treatment of PDGF-BB bulk drug substance
with carboxypeptidase B simplifies the RC CZE electropherogram and
allows quantification of the three isoforms of rPDGF-BB as defined
by the clipping between Arg-32 and Thr-33 (the isoforms referred to
as intact, single-clipped, and double-clipped).
[0050] The RC CZE method of analysis of PDGF-BB, as modified in the
present invention to isolate intact, single-clipped, and
double-clipped by use of carboxypeptidase B, is useful, in a
quality control context, for analysis of a bulk drug substance
comprising the rPDGF-BB isoforms described herein. The advantage of
use of RC CZE for analyzing PDGF-BB in a quality control context is
the ease and precision achieved by this method, which makes the
method an appropriate method for such routine activities of
analysis as required in quality control of bulk drug
substances.
[0051] Thus the methods of the invention are useful in analyzing
and separating intact, single-clipped, and double-clipped isoforms
of rPDGF-BB. Following separation of the desired intact,
single-clipped, or mixture of intact and single-clipped isoforms of
rPDGF-BB, contaminating buffer or other impurities can be removed
where necessary to produce compositions comprising substantially
purified isoforms of interest. These compositions can be prepared
for use as a therapeutic agent as described below. By "therapeutic
agent" is intended an agent that accomplishes a therapeutic goal,
as for example, healing a wound.
[0052] Compositions comprising the substantially purified intact,
single-clipped, or a mixture of intact and single-clipped isoforms
of rPDGF-BB are useful as therapeutic agents in the treatment of
wounds, particularly those wounds that are normally repaired in
response to the release of the native PDGF.
[0053] The release of PDGF in vivo acts as a chemotactic agent to
attract monocytes, fibroblasts, and smooth muscle cells to the site
of injury, and to initiate cell replication for the purposes of
wound repair. This growth factor has been shown to promote healing
of incision wounds in rats and healing in regions of the body such
as chambers or subcutaneous spaces. Thus PDGF has application in
healing such wounds as ulcers, bums, and skin grafts. In addition,
PDGF can be used to heal wounds resulting from damaged connective
tissue, the repair of which requires fibroblast proliferation and
collagen deposition. In addition, where wound healing is retarded
by any number of factors including, but not limited to advanced
age, diabetes, cancer, and treatment with anti-inflammatory drugs
or anticoagulants, PDGF can be used to promote normal wound healing
in mammals in situations that would otherwise delay wound
healing.
[0054] The usefulness of platelet-derived growth factor as a
therapeutic agent for full and partial thickness bum repair has
also been described by Danilenko et al. (1995) Am. J. Pathol.
147:1261-1277. In this manner, recombinant PDGF-BB induces marked
extracellular matrix and granulation tissue production such that
the burn defects are repaired.
[0055] In the context of diabetes, a condition with the
complication of delayed wound healing, it has been postulated that
refractory diabetic wounds are the result of deficiencies in growth
factors and/or cytokines that are important for the healing
process. Some preliminary studies have shown that application of
certain growth factors or cytokines can facilitate wound healing,
as described in Doxey et al. (1995) Life Sciences 57(11):111-23. In
this study, PDGF of wounded diabetic rats was produced at low
levels compared to control animals. These rats had a delayed
production of PDGF in cells surrounding the wounds that occurred
many days after the wound was incurred, possibly explaining the
delay in wound healing in the diabetic animals. Thus, there is a
potential application for administration of PDGF-BB to diabetics to
induce and normalize wound healing.
[0056] Mechanisms of action of PDGF-BB and the other PDGF dimers,
including PDGF-AA and PDGF-AB, are discussed generally in Lepisto
et al. (1995) Biochem. Biophys. Res. Comm. 209(2):393-9. The dimers
PDGF-AA and PDGF-BB down-regulate both the steady-state levels of
pro-alpha 1 (I), and pro alpha 1 (III) collagen chain mRNAs and the
production of collagen, both in a dose-dependent manner.
[0057] Additionally, compositions comprising PDGF can facilitate
the healing of injured or depleted bones by promoting growth of the
connective tissue, growth of the bone and cementum, and by
stimulation of protein and collagen synthesis in the region of the
injured bone, especially as in the case, for example, of
periodontal disease (see WO 88/03409).
[0058] Compositions comprising PDGF can provide fast, effective
healing for such wounds as, for example, bedsores, lacerations, and
burns, as described in WO 91/15231 and U.S. Pat. No. 5,019,559,
wherein PDGF in a composition with insulin-like growth factor (IGF)
promotes an increase in new connective tissue and an increase in
the growth of epithelial tissue.
[0059] Compositions comprising PDGF can be administered in a gel
formulation, and such a formulation is suitable for topical
administration to promote wound healing in conditions such as
ulcers, superficial wounds and lacerations, abrasions, surgical
wounds, burns, and bone defects, as described in WO 93/08825.
[0060] For use as a therapeutic agent for the treatment of wounds,
compositions comprising the intact, single-clipped, or a mixture of
intact and single-clipped isoforms of PDGF-BB described herein can
be administered topically with an appropriate pharmaceutically
acceptable carrier or in a substantially purified preparation of
protein such that it is free from impurities and elements that
would interfere with a therapeutic use. Such preparations that are
free of toxic, antigenic, inflammatory, or other deleterious
substances and that are at least about 80% pure are considered
appropriate. The concentration of the protein can be from about 1
to about 25 .mu.g/ml of total volume, or can be in a range as broad
as about 1 ng/ml to about 50 .mu.g/ml depending on the therapeutic
use for the rPDGF-BB. A therapeutically effective amount sufficient
to accelerate the rate of appearance and increase the number of new
fibroblasts in the wound space and to stimulate DNA synthesis in
and collagen deposition by those fibroblasts will be in the range
of about 1 to about 5 milliliters of the preparation, depending on
the characteristics of the wound.
[0061] A pharmaceutical composition comprising substantially
purified intact or single-clipped isoforms of rPDGF-BB or a mixture
of such isoforms and an appropriate pharmaceutically acceptable
carrier can be formulated for administration as a therapeutic agent
for the treatment of wounds. Pharmaceutically acceptable carriers
may be large, slowly metabolized macromolecules such as proteins,
polysaccharides, polylactic acids, polyglycolic acids, polymeric
amino acids, amino acid copolymers, and inactive viruses in
particles. Such carriers are well known to those of ordinary skill
in the art. Pharmaceutically acceptable salts can be used therein,
for example, mineral acid salts such as hydrochlorides,
hydrobromides, phosphates, sulfates, and the like; and the salts of
organic acids such as acetates, propionates, malonates, benzoates,
and the like. Pharmaceutically acceptable carriers in therapeutic
compositions may contain liquids such as water, saline, glycerol,
and ethanol. Additionally, auxiliary substances, such as wetting or
emulsifying agents, pH buffering substances, and the like, may be
present in such vehicles.
[0062] The method for formulating a pharmaceutical composition is
generally known in the art. A thorough discussion of formulation
and selection of pharmaceutically acceptable carriers, stabilizers,
and isomolytes can be found in Remington's Pharmaceutical Sciences,
18.sup.th edition (Mack Pub. Co., Eaton, Pa., 1990).
[0063] Typically, the therapeutic pharmaceutical compositions are
prepared as injectables, either as liquid solutions or suspensions.
Solid forms suitable for solution in, or suspension in, liquid
vehicles prior to injection may also be prepared. Liposomes are
included within the definition of a pharmaceutically acceptable
carrier. Some liposome compositions are described in U.S. Pat. No.
5,422,120; WO 95/13796; WO 94/23697; WO 91/14445; and EP 524,968
B1.
[0064] Administration of the PDGF-BB therapeutic pharmaceutical
composition can be by any means and to any location of the body of
the animal being treated as is appropriate for the wound being
treated. Thus, the PDGF-BB can be administered, for example,
parenterally or topically. Parenteral administration can include
subcutaneous, intravenous, intra-arterial, intramuscular, or direct
injection into a region or organ of the body.
[0065] The following examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
Example 1
Preparation of PDGF-BB Bulk Drug Substance
[0066] Recombinant human PDGF-BB (rhPDGF) was produced by a strain
of the yeast Saccharomyces cerevisiae genetically modified with a
multicopy yeast expression plasmid that includes a gene encoding
the mature human PDGF-B polypeptide chain. Details of this yeast
expression system are disclosed in U.S. Pat. No. 6,017,731, and in
the copending application entitled "Method for Expression of
Heterologous Proteins in Yeast," Ser. No. 09/340,250; herein
incorporated by reference.
[0067] The S. cerevisiae strain MB2-1 was developed to contain the
genotype: Mat.alpha., ura3), leu2-3, leu2-112, his3-11, his3-15,
pep4), [cir.sup.o]. It is auxotrophic for uracil, leucine and
histidine, requiring these nutritional supplements when grown in
minimal medium. MB2-1 does not contain an endogenous 2-.mu.
plasmid, which tends to interfere with the stability of the
introduced plasmids and encourages recombination between endogenous
and introduced plasmids. The strain does not express functional
protease A, the product of the PEP4 gene, which interferes with the
production of heterologous proteins. MB2-1 was designed to impart
these favorable characteristics, which include selection for high
expression of heterologous proteins.
[0068] The vector selected for expressing rhPDGF-BB, pAB24, is a
yeast-bacteria shuttle vector. The plasmid is a chimera of
sequences from pBR322, derived from several naturally occurring
bacterial plasmids, and sequences of the endogenous S. cerevisiae
2-.mu. plasmid (Broach (1981) in Molecular Biology of the Yeast
Saccharomyces (Cold Spring Harbor Press, New York), 1:445-470).
Characteristics of this vector are described in detail in U.S. Pat.
No. 6,017,731, and in the copending patent application entitled
"Method for Expression of Heterologous Proteins in Yeast," Ser. No.
09/340,250; herein incorporated by reference.
[0069] An expression cassette comprising a nucleotide sequence
encoding rhPDGF-BB was ligated into the pAB24 vector to generate
the yeast expression plasmid pYL7PPB. Methods for constructing this
plasmid and a detailed description of its characteristics are
described in detail in U.S. Pat. No. 6,017,731, and in the
copending patent application entitled "Method for Expression of
Heterologous Proteins in Yeast," Ser. No. 09/340,250; herein
incorporated by reference.
[0070] Briefly, plasmid pYL7PPB includes an expression cassette
with the following features. Transcription initiation and
termination are mediated by the inducible hybrid yeast promoter
ADF/GAP and the Matx transcriptional terminator. The cassette
further includes an open reading frame encoding a truncated yeast
alpha-factor leader to mediate secretion of native human proPDGF-B.
The prosequence included in the expression construct is only the
native N-terminal propeptide encoding sequence; the native
C-terminal propeptide sequence was not included in the construct.
Inclusion of the N-terminal propeptide resulted in enhanced
expression of rhPDGF-BB, as disclosed in copending application Ser.
No. 08/989,251. Dibasic processing sites at the truncated alpha
factor leader/N-terminal propeptide and N-terminal
propeptide/PDGF-B junctions were included to facilitate production
of correctly processed rhPDGF-B polypeptide by yeast. The
nucleotide and predicted amino acid sequence of open reading frame
from pYL7PPB encoding truncated alpha factor leader--proPDGF-B are
disclosed in U.S. Pat. No. 6,017,731, and in copending application
Ser. No. 09/40,250.
[0071] Isolation of the cDNA encoding HPDGF-B was performed using a
cDNA library prepared from RNA isolated from a human clonal glioma
cell line. The isolation and characteristics of this cDNA have been
described in Westermark et al. (1982) J. Neurosci. Res. 8:491 and
Ponten and Westermark (1978) Med. Biol. 56: 184 (1978). This cell
line was utilized as a source of RNA for PDGF gene isolation
because it had been shown to produce higher levels of PDGF receptor
competing activity than other cell lines tested.
Example 2
Enrichment from rhPDGF-BB Bulk Drug Substance of Intact and Single
Clipped rhPDGF-BB Isoforms Using TSK Sulfopropyl Cation Exchange
Chromatography
[0072] Making of the rhPDGF-BB bulk drug substance is described in
Example 1. The overall goal of this procedure was to generate
milligram quantities of intact, single-clipped, and double-clipped
isoforms of rhPDGF-BB using repetitive TSK SP 5PW (Tosoh-Haas)
chromatography of pure rhPDGF-BB produced in a yeast host cell,
then to determine the relative biological activity of the three
isoforms using the mitogen assay. In the process, it was shown that
when, instead of the 0 to 1 M NaCl called for in the manufacturing
protocol, SP TSK 5PW was eluted with a 0 M to 0.6 M NaCl gradient,
a significant portion of the double-clipped isoform of rhPDGF-BB
appeared to elute at the front of the gradient, separated away from
the rest of the material. As the double-clipped isoform is the
least biologically active (see examples below), it could be useful
to incorporate this simple change (to the 0-0.6 M gradient) in a
future, large-scale second-generation process.
[0073] The column was prepared by packing 50 ml of TSK SP 5PW
(supplied pre-swollen) into an empty Pharmacia column K26
(2.5.times.10 cm). The packed column was washed with not less than
5 column volumes (c.v.) of loading buffer (0.05 M Na Acetate, pH
4.5). rhPDGF-BB produced by the method of Example 1 was applied,
and the column washed with not less than 2 c.v. loading buffer, and
then 2 c.v. 0.05 M Na Phosphate pH 7 (with NaOH). The product was
eluted with a 10 c.v. linear gradient (from 0 to 0.6 M) NaCl
(buffer B) in 50 mM sodium phosphate pH 7.0. The elution peak was
fractionated.
[0074] In the runs shown here, starting material was a final pool
of rhPDGF-BB as described in Example 1. The material was in Na
Phosphate at pH 6 at 9.9 mg/ml; the pH was adjusted to 4.5 with 1 M
NaH.sub.3PO.sub.4. The concentration of rh-PDGF-BB was 9 mg/ml
after pH adjustment, and conductivity of the load was 1.62 mS/cm at
25.degree. C. All work was done at room temperature. 477 mg (53 ml)
was loaded over the column at 2 ml/min. The column was washed with
2 c.v. of loading buffer (100 ml), and then the 0-0.6 M NaCl
gradient was applied in approximately 10 c.v. (492 ml). 15 ml
fractions were collected, and fractions were analyzed by PAGE
(10-20% Novex Tricine Gels), and absorbencies measure. The protein
eluted beginning at fraction 28 through about fraction 50. Based on
the gels, fractions 28-30 were pooled, diluted 1:1 with distilled
water (to get the conductivity down low enough to permit binding),
reapplied to the SP column, and eluted under the same conditions.
When analyzed by mitogenic assay, the load was shown to have 14% of
the activity of the reference standard, indicating that these
fractions represented a large amount of the double-clipped isoform.
However, by SDS PAGE gels sufficient analysis could not be made as
to quantity of PDGF-BB dimeric isoform. Analysis of these fractions
by the RC CZE method described in Example 4 indicates the extent of
the purity of the fractions and aids in making an assessment of
which fractions contain which isoform and in which relative
amounts. By A.sub.280 the pooled tubes (28-30) were shown to be
about 69 mg, or 15% of the 477 mg loaded on for the run. The
results of the mitogenic assay conducted with this material
indicates low activity and a double-clipped isoform. Analysis of
these fractions using RC CZE would then be appropriate for a final
determination of the several isoforms isolated, and a
quantification of the amounts.
Example 3
High Temperature Linear Gradient RP HPLC Analysis of Nonreduced
rhPDGF-BB
[0075] The bulk drug substance rhPDGF-BB produced in yeast as
described in Example 1 was analyzed on a 5 micron, 300 Angstrom,
Delta-Pak C3 (3.9 mmID.times.15 cm L) column using a Spectra
Physics 8800 solvent delivery pump equipped with a WISP 712
autoinjecter and a PE 135 diode array detector. Column temperature
was controlled by the use of a Nesslab glycol cooling bath of a
Jones Chromatography heating block. The separation was achieved at
a 1.2 ml/min flow rate using a 5 to 60% acetonitrile/0.1% TFA
linear gradient over 40 minutes.
[0076] When rhPDGF-BB was analyzed at 5.degree. C., six distinct
peaks with several smaller shoulders were resolved. As the
temperature increased (5.degree. C., 20.degree. C., 33.degree. C.,
60.degree. C.), the profile became less defined. However, at
60.degree. C. the peak became more resolved although shoulders were
still present. The early-eluting doublet peak (24 minutes) observed
in the 5.degree. C. profile disappeared as the temperature was
increased. To further characterize the distinct peaks generated at
5.degree. C., the rhPDGF-BB was semi-prepartively fractionated.
These fractions were analyzed by RP HPLC and each fraction
rechromatographed, resulting in a profile that displayed
significant amounts of protein present in other fractions. Thus, it
is apparent that a slow but reversible equilibrium exists between
isoforms separated at 5.degree. C. resulting from the
interconversion of rhPDGF-BB conformers. Isomerization of the
Pro-Pro bond is probably responsible for the observed slow
equilibrium among rhPDGF-BB conformers.
[0077] The RP HPLC analysis of nonreduced rhPDGF-BB was then
performed using a Zorbax 300SB-C18 reversed-phase column (4.6 mm
ID.times.25 cm L) maintained at 85.degree. C. using a linear
TFA/acetonitrile gradient. This method resolved the 21 predicted
clipped and truncated isoforms of rhPDGF-BB, which are clustered
into three major groups (intact, single-clipped, and
double-clipped). Fractions collected across each group were
analyzed using RP HPLC of reduced rhPDGF-BB. The results indicated
that the multiple peaks in each group are due to different levels
of C-terminal truncation of Arg-32 and Thr-109. Further attempts to
improve the separation of clipped and intact isoform from these
three groups were unsuccessful using a linear gradient. Thus, high
temperature at a linear gradient of RP HPLC was able to resolve the
heterogeneity resulting from post-translational modifications due
to clipping and truncation occurring on both faces of the molecule,
and the phenomena of multiple Pro-Pro conformers was not observed
at high temperature.
[0078] A combination isocratic/step gradient was then employed in
addition to the high temperature to improve separation of the
isoforms. The high temperature step gradient resolved the
double-clipped isoform at 29% acetonitrile, the single-clipped
isoform at 30% acetonitrile, and the intact isoform at 33%
acetonitrile. Unlike the linear gradient above, isoforms due to
truncation are not resolved, and thus simplify the profile greatly.
Accordingly, separation of the isoforms of rhPDGF-BB predominantly
on the basis of clipping was achieved by the high temperature
combination isocratic/step gradient just described, therefore
simplifying the chromatographic profile greatly, and facilitating
analysis and separation of key functional isoforms of
rhPDGF-BB.
Example 4
Reverse Charge Capillary Zone Electrophoresis Assay for rhPDGF-BB
Including Pretreatment with Carboxypeptidase B
[0079] A sample of rhPDGF-BB recombinantly produced in yeast as
described in Example 1 and a reference material having a known
composition of PDGF-BB were assayed using reverse charge capillary
zone electrophoresis (RC CZE). The samples of interest were diluted
in running buffer having Tris buffer at pH 8.0 to a concentration
of 1.0 mg/ml as determined by spectrophotometry. 100 .mu.l of 25
U/ml carboxypeptidase B was added to 50 .mu.l of test sample. After
30 minutes of incubation at room temperature, 25 pI of 0.1N HCl was
added. A sequence table was prepared on a Turbochrom Sequence
Screen. Degas reagents necessary for carrying out the assay were
prepared by placing 1500 .mu.l of each of the following into a
separate 1700 .mu.l polypropylene microfuge tube: 1N NaOH, eCAP.TM.
Regenerator solution, and 50 mM Tris buffer (pH 8.0). The tubes
were microfuged for 5 minutes at the highest rpm setting
available.
[0080] The assay was run on a capillary electrophoresis system
(BioFocus.TM. 3000) configured with the following information: prep
1: preinject NaOH, 180 sec; prep 2: preinject AMREGEN, 180 sec;
prep 3: preinject AMRINSE 180 sec; pressure: 2 psi*sec; polarity:
neg to pos; voltage: 15.00 kV; current limit: 50.00 uA; inlet
buffer: AMRUN; outlet buffer: AMRUN; cartridge temp: 25 degrees
Celsius; run time: 10 min; single wavelength programmable detector
mode; and lamda(nm): 214. The samples were loaded into the
BioFocus.TM. 3000. Enable Peak detection was set prior to rise in
the electropherogram before the double-clipped peak and Disable
Peak detection was set after the horizontal line following the
intact rhPDGF-BB peak.The electopherogram was acceptable if the
difference in duplicate sample measurements was less than 10%.
[0081] The results of analysis of clinical lots of rhPDGF-BB by RC
CZE indicated the following compositions of intact, single-clipped,
and double-clipped isoforms for material intended for commercial
use: TABLE-US-00001 batch double-clipped single-clipped intact 1
47% 31% 22% 2 44% 33% 23% 3 45% 33% 22% 4 46% 33% 21% 5 45% 33% 22%
6 45% 32% 23%
Example 5
Mitogenic activity of Single-clipped, Double-clipped and Intact
Isoforms of rhPDGF-BB
[0082] The ability of PDGF to stimulate DNA synthesis and cell
growth in culture can be tested by .sup.3H-Thymidine incorporation
into DNA of cultured cells responsive to PDGF,5 and results in the
demonstration of biological activity of PDGF or PDGF-like
molecules. Such an assay is described in U.S. Pat. No. 4,849,407,
and in Cook et al. (1992) Biochem. J. 281:57-65, herein
incorporated by reference. This is a standard assay for determining
potential therapeutic benefit of PDGF.
[0083] The mitogenic assay for PDGF, a mitogen, is believed in the
art to represent potential usefulness as a pharmaceutical
composition. It has been determined previously that the C-terminal
truncations do not have any effect on the biological activity. It
has also determined that over the narrow range of peak elution, the
acetonitrile and triflouroacetic acid (ACN-TFA) components of the
chromatography had a consistent effect on the mitogenic assay.
[0084] Purified single-clipped, double-clipped and intact forms of
rhPDGF-BB were collected through repeated cycles of high
temperature nonreduced RP HPLC. The single-clipped, double-clipped
and intact forms were tested using the high temperature nonreduced
and reduced RP HPLC assays, and the purities were found to be 95%,
97%, and 95%, respectively. Samples of each form were then tested
by the mitogenic activity assay. All samples were assayed in
triplicate in the same assay. The fractions of each isoform were
collected, and it was determined that the single-clipped isoform
had 120% activity, the intact isoform had 100% activity, and the
double clipped had 20% activity. The reference sample (an
unpurified cell culture comprising a mixture of intact,
single-clipped, and double-clipped isoforms of rhPDGF-BB) exhibited
50% activity. This indicated that it may be desirable to remove the
double-clipped isoform in a rhPDGF-BB bulk substance that is
intended for therapeutic use.
Example 6
PDGF-BB to Treat a Wound
[0085] A PDGF-BB mixture of intact and single-clipped isoforms is
prepared in a topical cream. Application of about 50 to about 100
mM of PDGF-BB mixture is made to a third degree burn wound from 1
to 3 times daily for a month, or until the wound is healed.
Standard application procedures for dressing burn wounds are
practiced for the administration of the PDGF-BB cream to the burn
wound.
[0086] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0087] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
* * * * *