Note: Descriptions are shown in the official language in which they were submitted.
- l 1337671
PROCESS FOR RECOVERING PURIFIED, OXIDIZED,
RENATURED RECOMBINANT INTERLEUKIN-2
FROM MICROORGANISMS
Description
Technical Field
The invention is in the field of biochemistry
and relates to a process for recovering purified,
renatured recombinant interleukin-2 (IL-2) from the
microorganisms in which it is produced.
Backqround
IL-2, a lymphokine which is produced by normal
peripheral blood lymphocytes and induces proliferation
of antigen or mitogen stimulated T cells after exposure
to plant lectins, antigens, or other stimuli, was first
described by Morgan, D.A., et al, Science (1976)
193:1007-1008. Then called T cell growth factor because
of its ability to induce proliferation of stimulated T
lymphocytes, it is now recognized that in addition to
its growth factor properties it modulates a variety of
functions of immune system cells in vitro and in vivo
and has been renamed IL-2. IL-2 is one of several
1337~71
--2--
lymphocyte-produced messenger-regulatory molecules that
mediate immunocyte interactions and functions.
IL-2 was initially made by cultivating human
peripheral blood lymphocytes (PBL) or other IL-2-
producing cell lines. See, for instance, U.S. Patent
No. 4,401,756. Recombinant DNA technology has provided
an alternative to PBLs and cell lines for producing
IL-2. Taniguchi, T., et al, Nature (1983) 302:305-310
and Devos, R., Nucleic Acids Research (1983) 11:4307-
4323 have reported cloning the human IL-2 gene and
expressing it in microorganisms.
Native human IL-2 is an antigen-nonspecific,
genetically unrestricted soluble factor produced by
erythrocyte rosette positive T cells stimulated with
antigens, mitogens or alloantigens. It is a protein
with a reported molecular weight in the approximate
range of 13,000 to 17,000 daltons (S. Gillis and J.
Watson, J Exp Med (1980) 159:1709) and an isoelectric
2 point in the approximate range of pH 6-8.5. Human IL-2
has a number of in vitro and in vivo effects including
enhancing the proliferative responses of human
peripheral blood mononuclear cells or murine thymocytes,
enhancing the immune response in humans and -in animals
against bacterial, parasitic, fungal, protozoan and
viral infections, and supporting the growth of
continuous T cell lines.
Human IL-2 has been obtained from genetically
engineered E. coli as an unglycosylated protein with
biological activities equivalent to those of native,
glycosylated IL-2. (Taniguchi et al, Nature (1983)
11:4307-4323; Rosenberg et al, Science (1984) 223:1412-
1415; Wang et al, Science (1984) 224:1431-1433; and
Doyle et al, J Biol Resp Modifiers (1985) 4:96-109).
Rosenberg and his coworkers have shown that systemic
- 133767l
administration of recombinant IL-2 in high doses causes
regression of established metastatic cancers in mice
(Rosenberg et al, J ExP Med (1985) 161:1169-1188); and,
in conjunction with lymphokine-activated killer cells
(Rosenberg et al, New Enq J Med (1985) 313:1485-1492)
and tumor-infiltration lymphocytes (Rosenberg et al,
Science (1986) 233:1318-1321), in humans.
U.S. Patent No. 4,518,584 discloses muteins
(analogs) of IL-2 in which the cysteine normally
occurring at position 125 of the wild-type or native
molecule has been replaced with a neutral amino acid,
such as serine or alanine. European Patent (EP)
publication 200,280 discloses muteins of IL-2 whereby
the methionine at position 104 has-been replaced by a
conservative amino acid.
Microbially produced IL-2 is not glycosylated
and is primarily produced in a denatured state. It is
largely insoluble and, when expressed at high levels, it
precipitates intracellularly in the form of "refractile"
or n inclusion" bodies which appear as bright spots
visible within the enclosure of the cell under a phase
contrast microscope at magnifications down to 1000 fold.
The problem addressed by the present invention is how to
efficiently recover the IL-2 from the cell in a
purified, cystine-bridged, renatured form that is
acceptable for clinical use.
The heretofore available methods for recover-
ing microbially produced IL-2 are described below.
U.S. Patent No. 4,569,790 describes a process
for recovering recombinant IL-2 from an IL-2-producing
microorganism in which the cell is disrupted, the
disruptate is extracted with an aqueous solution of a
chaotropic agent such as urea, the IL-2 is solubilized
with a surfactant, e.g., sodium dodecyl sulfate (SDS),
,
- 13~7671
--4--
and the IL-2 is separated in the presence of a reducing
agent.
Commonly owned U.S. Patents Nos. 4,530,787 and
4,572,978 described processes for purifying recombinant
IL-2 from microorganisms in which partially purified
reduced IL-2 is selectively oxidized under controlled
conditions to its oxidized (cystine) form. The former
patent uses o-iodosobenzoic acid as an oxidizing agent
and the latter uses Cu+2 cation as an oxidation
promoter.
European Patent publication 206,828 published
30 December 1986, and entitled "Process for Recovering
Refractile Bodies Containing Heterologous Proteins from
Microbial Hosts" disclose methods for recovering and
purifying refractile bodies of IL-2 from E. coli. To
isolate the refractile material, the processes initially
involve disrupting the cell wall and membrane of the
host cell, removing greater than 99% by weight of the
salts from the disruptate, redisrupting the desalted
disruptate, adding a material to the disruptate to
create a density or viscosity gradient in the liquid
within the disruptate, and separating the refractile
material from the cellular debris by -high-speed
centrifugation. The IL-2 is then solubilized with a
solubilizing agent such as SDS, chromatographed to
remove high molecular weight contaminants, oxidized, and
purified by a combination of HPLC, ultrafiltration and
gel filtration.
An abstract titled "Purification and Renatura-
tion of Recombinant Interleukin-2" presented at the 6th
International Symposium on HPLC of Proteins, Peptides
and Polynucleotides at ~8aden-Baden, West Germany in
October 1986 describes a process in which recombinant
IL-2 is solubilized from inclusion bodies with 6 M
13376~1
guanidine hydrochloride/10 mM dithiothreitol (DTT) and
purified in a reduced, denatured form by FPLC gel
permeation. The solution from the FPLC gel permeation
is diluted to effect renaturation and autooxidation. In
this regard U.S. Patents Nos. 4,511,502; 4,511,503;
4,512,922 and 4,518,526; and EP publication 114,506
describe a similar procedure for purifying heterologous
proteins in general from refractile bodies. In such
processes, the oxidation and renaturation of the IL-2
are carried out in a single step. However, because of
disparate solubility characteristics between the reduced
and oxidized forms of IL-2, it is difficult to achieve
high yields of renatured oxidized IL-2 in such a
process.
EP publication 145,390 describes a process for
recovering rIL-2 from E. coli in which the cells are
suspended in 7 M guanidine hydrochloride, solids are
removed by centrifugation, the rIL-2-containing super-
natant is dialyzed to remove the guanidine hydrochloride
and the dialyzate is purified by anion exchange
chromatography, gel filtration and RP-HPLC.
The present invention is directed to an
improved recombinant IL-2 purification process in which
the oxidation and renaturation are carried out in
distinct steps.
Disclosure of the Invention
The present invention relates to a high yield
process in which IL-2 is separated from a cellular
disruptate in the form of a refractile body, dissolved
with a chaotropic agent and oxidized and renatured in
separate steps followed b-y purification to a clinically
acceptable level.
1337671
--6--
More specifically, the invention relates to a
process for recovering purified soluble recombinant IL-2
from a transformed microorganism containing the IL-2
comprising:
(a) disrupting the cell membrane and cell wall
of the microorganism;
(b) separating water insoluble material from
the disruptate;
(c) mixing the insoluble material of step (b)
at a pH of about 7 to about 9 with an aqueous solution
of a reducing agent and a strongly denaturing
concentration of a chaotropic agent whereby the IL-2 in
the insoluble material is dissolved;
(d) precipitating the IL-2 out of the
IL-2-containing solution and recovering the precipitate;
(e) solubilizing the IL-2 precipitate;
(f) oxidizing the IL-2 in the solution whereby
the natural disulfide bridge of IL-2 is formed;
(g) after the oxidation of step (f) is
complete, reducing the concentration of chaotropic agent
in the solution to a level at which the oxidized IL-2 is
renaturated and a precipitate forms;
(h) separating the precipitate of step (g)
from the solution to provide a supernatant;
(i) purifying the oxidized, renatured IL-2 in
the supernatant by reverse-phase high performance liquid
chromatography followed by dissolution of the pool in a
solution of chaotropic agent and removal of the
chaotropic agent from the solution, or by hydrophobic
interaction chromatography combined with ion exchange
chromatography, or by ion exchange chromatography; and
(j) recovering ~a purified oxidized, soluble
heterologous human IL-2 composition having an IL-2
content of at least about 95% as determined by reducing
1337~71
-7-
sodium dodecyl sulfate polyacrylamide gel electro-
phoresis analysis, a solubility of at least 3 mg IL-2
per ml, a specific activity of at least 1 x 107 units/mg
as determined by HT-2 cell proliferation assay, and an
endotoxin content of less than about 0.1 nanograms per
mg of IL-2. Preferably the composition is also sub-
stantially free of pyrogens as determined by the U.S.P.
rabbit pyrogen test at a dosage of 1.0 x 103 units per
kg, more preferably 3.3 x 105 units per kg.
Brief Description of the Drawinqs
Figure 1 is a flow diagram of the preferred
embodiment of the invention process.
Figure 2 is a graph of the results of the
immunoassay described in Example 1, infra. The closed
circles represent the SDS-process IL-2 and the closed
squares represent the guanidine-process IL-2, both of
- which are described in the example.
Modes for Carryinq Out the Invention
A. Definitions
As used herein, the term "IL-2" refers to
recombinant interleukin-2 or interleukin-2-like
polypeptides produced by a transformed microorganism and
whose amino acid sequence is the same as or similar or
substantially homologous to the unglycosylated and/or
glycosylated native interleukin-2. Examples of such
recombinant IL-2s are those described in European
published patent applications 91,539, 88,195, and
109,748, as well as those described in U.S. Patent No.
4,518,584, copending, commonly assigned U.S. Serial No.
893,186, filed 5 August -1986, EP publication 200,280,
and bovine IL-2 as described by Cerretti et al, Proc
Natl Acad Sci USA (1986) 83:3223-3227.
~33~ 67 1
-8-
The recombinant IL-2s particularly preferred
herein are those biologically active muteins (analogs)
of human IL-2 in which amino acid residues not essential
to biological activity have been deliberately deleted in
some instances or replaced with a conservative amino
acid, as indicated below. More specifically, preferred
recombinant IL-2s include those wherein the cysteine
residue at position 125 is replaced with another amino
acid, preferably neutral or conservative, to eliminate
sites for intermolecular crosslinking or incorrect
intramolecular disulfide and, optionally, the N-terminal
alanine residue of the native counterpart is eliminated.
As used herein, such neutral or conservative amino acids
are glycine, serine, valine, alanine, leucine,
isoleucine, tyrosine and methionine. More particularly,
preferred recombinant IL-2 muteins in the formulations
of this invention are those wherein (1) the cysteine
residue at amino acid position 125 of the native
counterpart is replaced by a serine residue (designated
IL-2serl2s) or alanine residue (designated IL-2alal2s);
or (2) the initial alanine residue is eliminated and the
cysteine at position 125 is replaced by serine
(designated des-alanyl-IL-2serl2s)-
Other IL-2s particularly preferred herein are
those biologically active muteins described in European
Patent Publication 200,280 wherein oxidation-susceptible
methionine residues are replaced with a neutral or
conservative amino acid; a preferred mutein includes
replacing the methionine at position 104 with a
conservative amino acid such as alanine.
EP 200,280 also describes amino-terminal
deletions of IL-2 wherein one or more of the first six
-9- 1337671
amino acids are deleted. Preferred oxidation-resistant
muteins include alalo4serl2sIL-2, alalo4IL-2,
alalO4alal25IL-2, vallo4serl2sIL-2, vallo4IL-2,
vallo4alal2sIL-2, des-alalalal04serl2sIL-2, des-
alalalalo4IL-2, des-alalalal04alal2sIL-2, des-
alalvallo4serl2sIL-2, des-alalvallo4IL-2, des-
alalvallo4alal2sIL-2, des-alaldes-pro2alalo4serl25IL-2,
des-alal-des-pro2alal04IL-2, des-alaldes-
pr2alalO4alal25IL-2, des-alaldes-pro2vallo4serl2sIL-2,
des-alaldes-pro2-vall04IL-2, des-alaldes-
- Pro2vallo4alal25IL-2, des-alaldes-pro2des-
thr3alal04serl2sIL-2, des-alaldes-pro2des-
thr3alal04IL-2, des-alaldes-pro2-des-thr3alal04alal2sIL-
2, des-alaldes-pro2des-thr3-vallo4serl25IL-2~ des-
alaldes-pro2des-thr3vall04IL-2, des-alaldes-
pro2des-thr3vall04alal2sIL-2, des-alaldes-pro2des-
thr3des-ser4alal04serl2sIL-2, des-alaldes-pro2des-
thr3des-ser4alal04IL-2, des-alaldes-pro2des-thr3des-
ser4alal04alal2sIL-2, des-alaldes-pro2des-thr3des-
Ser4Val1045erl25IL-2, des-alaldes-pro2des-thr3de
ser4vall04IL-2, des-alaldes-pro2des-thr3des-
ser4vall04alal2sIL-2, des-alaldes-pro2des-thr3des-
ser4des-sersalalo4serl2sIL-2, des-alaldes-pro2des-
thr3des-ser4des-sersalal04IL-2, des-alaldes-pro2des-
thr3des-ser4des-sersalal04alal2s-IL-2, des-alaldes-
pro2des-thr3des-ser4des-sersvallo4-serl25IL-2~ des-
alaldes-pro2des-thr3des-ser4des-sers-vall04IL-2, des-
alaldes-pro2des-thr3des-ser4des-sers-vall04-alal2sIL-2,
des-alaldes-pro2des-thr3des-ser4-des-sersdes-
Ser6alalO4alal25IL-2, des-alaldes-pro2-des-thr3de5-
ser4des-sersdes-ser6alal04IL-2, des-alaldes-pro2des-
thr3des-ser4des-sersdes-ser6alalo4serl25IL-2~ des-
alaldes-pro2des-thr3des-ser4des-sers-des-
ser6vall04serl2sIL-2, des-alaldes-pro2des-thr3-des-
_
- 1337~71
--10--
.
sergdes-sersdes-ser6vall04IL-2, or des-alaldes-pro2des-
thr3des-ser4des-sersdes-ser6vallo4-alal25IL-2-
Other amino-terminal deletions of IL-2 are
disclosed in Chemical Abstracts (1987) 106:(21):170236f,
an abstract of Japanese Patent Publication No.
61/225199, published 6 October 1986, wherein any one of
the first 15 amino acids of IL-2 are deleted. PCT
87/04714, published 13 August 1987 describes deletions
or replacements of one or more of the amino acid
residues in positions 2 to 11 and/or 128 to 133 from the
amino-terminal alanine of IL-2.
The precise chemical structure of the IL-2
protein will depend on a number of factors. As
ionizable amino and carboxyl groups are present in the
molecule, a particular recombinant IL-2 protein may be
obtained as an acidic or basic salt, or in neutral form.
All such preparations which retain their activity when
placed in suitable environmental conditions are included
in the definition of IL-2 proteins herein. Further, the
primary amino acid sequence of the protein may be
augmented by derivatization using sugar moieties
(glycosylation) or by other supplementary molecules such
as lipids, phosphate, acetyl groups and the -like, more
commonly by conjugation with saccharides. Certain
aspects of such augmentation are accomplished through
post-translational processing systems of the producing
host; other such modifications may be introduced in
vitro. In any event, such modifications are included in
the definition of IL-2 protein herein so long as the
activity of the protein, as defined above, is not
destroyed. It is expected, of course, that such
modifications may quantitatively or qualitatively affect
biological activity, either by enhancing or diminishing
the activity of the protein in the various assays.
-11- 13~7S71
As used herein the term "transformed" in
describing host microorganism cell cultures denotes a
microorganism that has been genetically engineered to
produce an IL-2 polypeptide that is capable of
possessing the activity of native IL-2. Bacteria are
preferred microorganisms for producing the IL-2 protein.
E. coli is particularly preferred.
The term "chaotropic agent" refers to a
compound or compounds which, in aqueous solution and in
a suitable concentration, are capable of denaturing
recombinant IL-2. Correlatively the term "strongly
denaturing concentration" refers to a solution of a
chaotropic agent which will effectively "unfold" or
denature recombinant IL-2. Guanidine salts (e.g. the
hydrochloride) and alkali metal thiocyanate (e.g. sodium
thiocyanate) at concentrations in the range of about 4
to 9 M, preferably 6-9 M, are examples of chaotropic
agent solutions that will dissolve and denature
recombinant IL-2.
Cell Growth
The IL-2-producing transformed microorganisms
are grown in a suitable growth medium, typically to an
optical density (OD) of at least about 30 at 680 nm, and
preferably between about 20 and 40 at 680 nm. The
composition of the growth medium will depend upon the
particular microorganism involved. The medium is an
aqueous medium containing compounds that fulfill the
nutritional requirements of the microorganism. Growth
media will typically contain assimilable sources of
carbon and nitrogen, energy sources, magnesium,
potassium and sodium ions, and optionally amino acids
and purine and pyrimidine bases. (See Review of ~edical
Bioloqy, Lange Medical Publications, 14th Ed pp. 80-85
1~7671
-12-
(1980).) In expression vectors involving the trp
promoter, the tryptophan concentration in the medium is
carefully controlled to become limiting at the time
protein expression is desired. Growth media for E. coli
are well known in the art.
After the cells are harvested from the
culture, they may be concentrated, if necessary, to
about 20 to 150 mg/ml, preferably 80 to 100 mg/ml (OD 40
10to 300, preferably 160 to 200 at 680 nm) by cross-flow
filtration, centrifugation, or other conventional
methods.
Cell Disruption
15Following concentration of the harvested
culture, the cell membranes and cell walls of the
microorganisms are disrupted. Preferably a compound
which is non-toxic to humans, such as l-octanol, in an
amount of about 1% by weight of total components, is
added to the disrupted cells to ensure that no viable
recombinant organisms remain. Conventional cell
disruption techniques such as homogenization, sonica-
tion, or pressure cycling may be used in this step of
the process. The end point of the disruption step can
be determined by monitoring the optical density with the
absorbance at 260 nm of the suspension typically
increasing with cell lysis and by microscopic observa-
tion. In any event, the disruption should break
substantially all of the cells so that substantially no
intact cells are carried through to subsequent steps.
Treatment of DisruPtate to Isolate Insoluble IL-2 Bodies
After the cells have been disrupted, deionized
water is preferably added to the disruptate and greater
than 99% by weight of the salts are removed therefrom.
1337671
-13-
The salts are water-soluble materials composed of
oppositely charged small molecular weight ions. The
removal of these salts to reduce the ionic strength of
the disruptate may be accomplished by diafiltration
using deionized water to flush out the ions or by
centrifuging to pellet the cellular debris and
refractile bodies followed by resuspension in deionized
water. If diafiltration is employed, preferably
deionized water is continuously added such that the rate
of addition of watèr equals the filtration rate.
After the salts are essentially removed,
optionally a compound such as l-octanol may be added to
the desalted disruptate, if not added earlier, to ensure
that no viable recombinant organisms remain before
containment is broken. The desalted disruptate is again
disrupted as described above for the initial disruption.
After redisruption, density or viscosity is
increased and/or a gradient is created during
centrifugation in the liquid within the disruptate by
adding a material to the disruptate. There are several
means to accomplish this purpose, all relying on the
sedimentation characteristics of the particles by
varying the density and/or viscosity of the liquid
2 phase. One means to accomplish this goal is to add a
material which increases the density of the liquid to a
p of about 1.1 to 1.3 g/ml, preferably 1.13 to 1.17
g/ml.
Materials which may be used to accomplish this
density increase include a sugar or mixture of sugars,
such as, e.g., sucrose, dextrose, fructose, maltose,
maltotriose, and other mono-, dior polysaccharides. Most
preferably the sugar is sucrose. Alternatively, a
two-phase system of materials such as, e.g., a
glycerol/sucrose mixture may be used wherein the
13376~1
disrupted particles partition to the interface between
the heavy and light phases and can be eluted by a
liquid/liquid separation.
In addition, the viscosity of the liquid phase
may be increased to from 5 to 10 cps by any suitable
means such as by adding a viscous compound such as,
e.g., sucrose or glycerol thereto. Also, a gradient is
created if, e.g., the particles are in a 60% aqueous
glycerol suspension while the centrifuge bowl contains
80% aqueous glycerol.
The rL-2-containing refractile bodies are
separated from the cellular debris by high-speed
centrifugation. By "high-speed centrifugation" is meant
spinning the suspension in a centrifuge at about 8,000
to 40,000 times gravity (g), preferably about 10,000-
20,000 x g, for a suitable time period depending on the
volume, generally about 10 minutes to seventy-two hours.
The particle pellet or paste resulting from
the centrifugation contains approximately 15-70~ by
weight IL-2 as determined by SDS-polyacrylamide gel
electrophoresis and by Lowry assay (Lowry et al, J Biol
Chem (1951) 193-265-275)
-
The IL-2 in the particle paste or--pellet is
then dissolved and denatured by mixing the paste/pellet
with a solution of a strongly denaturing concentration
of the chaotropic agent and a reducing agent. The
chaotropic agent and reducing agent are in an aqueous
buffer at pH 7 to 9, preferably phosphate buffered
saline or Tris buffer. pH adjustment may be
accomplished by the addition of base such as NaOH. The
w/v ratio of pellet to solution will normally be in the
range of 0.01:1 to 0.25:I preferably 0.05:1 to 0.12:1.
Reducing agents that can be employed during the
dissolving/denaturing step include: B-mercaptoethanol,
- 1337~71
-15-
glutathione, cysteine and dithiothreitol (DTT). DTT is
the preferred reducing agent. The concentration of the
reducing agent in the medium will usually range between
about 10 to 100 mM with approximately 50 mM being the
preferred concentration. Chelating agents such as
ethylene diaminetetraacetic acid (EDTA) in concentra-
tions of 1 to 50 mM, preferably about 25 mM, and buffers
such as Tris-HCl at concentrations of 25 to 250 mM,
preferably 50 mM, may be included in the solution.
Elevated temperatures in the range of 35C to 50C,
- preferably about 40C, and a nitrogen blanket may be
used in this step. The dissolution/denaturation will
typically be complete after about 5 to 15 min of mixing.
After this time, the mixture may be centrifuged, pre-
ferably at 2000 x g to 4000 x g for about 10 to 30 min
to remove any undissolved materials.
The denatured IL-2 is next subjected to a
controlled oxidation. The reducing agent, along with
other contaminating material, is first removed using gel
filtration, diafiltration, or precipitation. Gels that
are capable of removing the reducing agent from the
protein solution are commercially available. For
instance, when DTT is used as a reducing agent Sephadex~
G-10, G-25, and G-50 gels may be used. The gel filtra-
tion will be run in a solution of the chaotropic agent
that maintains the protein in solution. When guanidine
hydrochloride is used, concentrations above about 6 M
are required to keep the IL-2 in solution and avoid
precipitate formation. After removing the reducing
agent the protein solution is diluted, if necessary,
with the solution of chaotropic agent to a protein
concentration of about 0.1 to 2 mg/ml, preferably about
0.25 to 1.0 mg/ml.
1337671
-16-
A preferred method of removing the reducing
agent along with contaminating material employs pre-
cipitation techniques. The reduced IL-2 is diluted to
approximately 2-30 mg of IL-2 per ml, preferably 5-10 mg
of IL-2 per ml with 7.0 M guanidine. This is then
diluted to approximately 1-5 M guanidine, preferably 3.5
M guanidine, and allowed to stand until the
precipitation is complete, generally two hours. Once
the precipitate begins to settle, the IL-2 can be
pelleted by centrifugation. Residual DTT may be removed
by washing the pellet with a buffer, preferably using a
2-4 M guanidine-containing buffer or a 1-2% polysorbate
80-containing buffer.
Preferred selective oxidation procedures are
described in commonly owned U.S. Patents Nos. 4,572,798
(using an oxidation promoter containing a Cu+2 cation
such as from CuC12, Cu(NO3)2, etc) and 4,~30,787 (using
o-iodosobenzoic acid) The Cu+2 oxidation
comprises reacting the aqueous solution of denatured
IL-2 at a pH between about 5.5 and 9, preferably 6 to 8,
and most preferably about 7.5, in the presence of air
with at least an effective amount of an~ oxidation
promoter containing a Cu+2 cation. Controlled oxidation
causes the formation of disulfide bridging in the IL-2
which conforms to the bridging in native IL-2 with no or
minimal overoxidation and formation of nonconforming
bridging or oligomers. Such oxidation enables the
production of high yields of the recombinant IL-2 with
the proper disulfide bridging.
The amount of oxidant or oxidation promoter
employed is at least an effective amount for oxidation,
i.e., an amount which at minimum will be necessary to
conduct the oxidation reaction effectively within a
-17- 1337~71
convenient period of time. An effective amount is the
amount approximately equivalent to the concentration of
free sulfhydryl groups in the IL-2 which are destined to
be involved in forming the desired disulfide bonds.
Preferably, the amount of CuC12 will range from about 5
to 275 micromolar. In the case of Q-iodosobenzoic acid
the mole ratio of oxidant to IL-2 will preferably be in
the range of about 0.05:1 to about 5:1, most preferably
about 0.8:1 to about 1:2. The concentration of IL-2 in
the reaction mixture is kept low, i.e., generally less
than about 5 mg/ml, preferably about 0.05 to about 2
mg/ml, and more preferably about 0.1 to about l mg/ml,
to reduce the likelihood of oligomer formation. The pH
is maintained between 5.5 and 9, preferably between 7
and 8 in the _-iodosobenzoic acid oxidation.
The reduced IL-2 must remain in solution for
effective oxidation to occur. Therefore, the reaction
mixture must contain a sufficient concentration of
chaotropic agent to keep the reduced IL-2 in solution.
As indicated above, when guanidine hydrochloride is
used, its concentration must be above 6 M. At such
concentrations, a substantial amount of the IL-2 will be
in a denatured form. For this reason, it is difficult
in the case of IL-2 to carry out the oxidation and
renaturation simultaneously and obtain high yields of
renatured I L-2 .
The temperature used in the oxidation will
normally be between about 20C and 40C, conveniently
room temperature. For Cu+2 oxidation, increasing the
reaction temperature increases the rate of reaction. The
oxidation reaction may be effectively terminated by,
e.g., lowering the pH to~a level at which the reaction
ceases, freezing the solution, or adding chelators such
1337671
-18-
as EDTA to the reaction mixture. Oxidation time will
normally be in the range of 4 hr to about one day.
When the oxidation is complete, the
concentration of the chaotropic agent (guanidine
hydrochloride) is reduced using dilution, dialysis, or
diafiltration, to a level which permits the oxidized
IL-2 to renaturate and refold into the configuration of
native IL-2. Phosphate buffer or Citrate buffer, 10 to
100 mM, preferably about 10 mM; NaCl, 10-150 mM,
preferably 40 mM and sucrose, 1-5%, preferably 2.5%,
are preferred diluents. Preferably, the IL-2 is
concentrated using an ultrafiltration membrane to avoid
handling large volumes of solution. The concentration
of guanidine hydrochloride agent is normally diluted or
diafiltered to below about 2 M, preferably below about
0.5 M. The dilution will typically be carried out at
about 4C to 25C. At such temperatures and reduced
guanidine hydrochloride concentration a precipitate of
extraneous host protein forms. This precipitate is
removed by filtration or centrifuging to provide a
supernatant containing the oxidized, renaturated IL-2.
The renatured, oxidized IL-2 is then purified
to remove endotoxins to a level that meets clinical
specifications (i.e., less than about 0.1 ng endotoxin
per ml of IL-2). The IL-2 is also preferably purified
to remove pyrogen so as to be substantially free of
pyrogens as measured by the U.S.P. rabbit pyrogen test
at a dosage of 1.0 x 103 units/kg, preferably 3.3 x 105
units/kg). The purification may be achieved by ion
exchange chromatography, or a combination of hydrophobic
interaction and ion exchange chromatography, or by
RP-HPLC.
The solution is then flowed over an ion
exchange column, such as a DEAE agarose column (e.g.,
... ~,
1337671
-19-
Pharmacia Fast-Flow Sepharose* DEAE). IL-2 is recovered
from the column with 10 mM citrate, pH 6.5. The eluted
IL-2 fractions may subsequently be loaded onto another
ion exchange column such as a carboxymethyl agarose
column (e.g., Pharmacia Fast-Flow Sepharose CM) that
binds IL-2 at a pH of 6 to 7.5. The bound IL-2 may be
eluted with an increasing salt gradient. The desired
IL-2 elutes at approximately 150 mM salt with the lower
isoelectric point forms of the protein eluting at lower
salt concentrations.
In the hydrophobic interaction/ion exchange
chromatography technique, (NH4)2SO4 is added to the IL-2
solution to a concentration of at least about 1.0 M,
preferably about 1.25 M. The solution is then loaded
onto a hydrophobic interaction column, such as a phenyl
agarose column (e.g., Pharmacia Phenyl Fast-Flow
Sepharose column). Bound IL-2 is recovered from the
column with a decreasing (NH4)2SO4 gradient, with the
IL-2 being collected in the fractions eluting at about
0.95 to 0.75 M (NH4)2SO4. Species of IL-2 and other
impurities (bacterial host proteins) having lower
isoelectric points than native IL-2 are then removed by
cation exchange chromatography using an exch-anger that
binds IL-2 at a pH of 6 to 7.5. A carboxymethyl agarose
column (e.g., Pharmacia Fast-Flow Sepharose CM) is a
preferred preparative cation exchanger. The solution is
contacted with the exchanger at the indicated pH range
and the IL-2 is eluted from the exchanger using an ionic
gradient. The desired IL-2 elutes at approximately 0.15
M salt with the lower isoelectric point forms of the
protein eluting at lower salt concentrations.
The HPLC puriflcation of the renatured IL-2
may be carried out in essentially the same manner as
described in U.S. 4,569,790 followed by redissolution in
*Trade maEk
13~7~71
-20-
a chaotropic agent and dialysis. Briefly, the solution
of IL-2 is chromatographed, precipitated, and the
resulting precipitate is taken up in the chaotropic
agent solution. The chaotropic agent is then removed by
dialysis or diafiltration. The IL-2 may be further
purified by cation exchange chromatography.
The purity of the renatured, oxidized IL-2
after the chromatography steps is at least about 95% and
usually at least about 98%, as determined by reducing
sodium dodecyl sulfate polyacrylamide gel electro
phoresis (SDS-PAGE) analysis. This pure IL-2 has a
solubility in PBS of at least about 5 mg/ml, a specific
activity of at least about 1 x 107 units/mg, usually 5 x
106 to 2 x 107 units/mg as determined by the HT-2 cell
proliferation assay, and endotoxin content of less than
about 0.1 ng/mg of IL-2. Also, preferably the IL-2 is
substantially free of pyrogens as determined by the
U.S.P. rabbit pyrogen test at a dosage of 1.0 x 103
units/kg, more preferably 3.3 x 105 units/kg.
Formulation
The purified IL-2 is rendered aqueous, its
concentration is adjusted, if necessary, to-0.01 to 2
mg/ml, and a water-soluble carrier is added to the
desired level. The carrier will typically be added such
that it is present in the solution at about 1% to 10% by
weight, preferably about 5% by weight. The exact amount
of carrier added is not critical. Conventional solid
bulking agents that are used in pharmaceutical tablet
formulation may be used as the carrier. These materials
are water soluble, do not react with the IL-2, and are
themselves stable. They are also preferably non-
sensitive to water (i.e., nonhygroscopic). Specific
examples of carriers that may be added include dextrose,
... ~, .
1337671
lactose, mannitol, sucrose, and other reduced sugars
such as sorbitol, starches and starch hydrolysates
derived from wheat, corn, rice, and potato,
microcrystalline celluloses, and albumin such as human
serum albumin. Mannitol, sucrose, and dextrose are
preferred.
The carrier adds bulk to the formulation such
that when unit dosage amounts of the solution are
lyophilized in containers, such as sterile vials, the
freeze-dried residue will be clearly discernible to the
naked eye. In this regard the preferred carrier,
mannitol, yields an aesthetically acceptable (white,
crystalline) residue that is not sensitive to water. The
nonsensitivity of mannitol to water may enhance the
stability of the formulation.
EP publication 215,658, published 25 March
1987, entitled "An Improved Formulation for Lipophilic
Proteins" (Hanisch et al) outlines an improved process
for recovering and purifying lipophilic recombinant
proteins such as IL-2 from microorganisms to yield a
protein preparation which may be formulated into a
stable pharmaceutical composition. Such a composition
carrying a therapeutically effective amount of the
biologically active recombinant lipophilic protein
dissolved in a non-toxic, insert, therapeutically
compatible aqueous-based carrier medium at a pH of 6.8
to 7.8 also contains a stabilizer for the protein, such
as human serum albumin, normal serum albumin and human
plasma protein fraction. The formulation aspects of
said EP publication 215,658 are herein incorporated by
reference as an alternative formulation route for the
purified IL-2. EP publication 215,658 outlines a low pH
formulation process. U.S. Patent No. 4,462,940 to
Hanisch et al, outlines a high pH formulation process,
I33767I
and the formulation aspects thereof are also herein
incorporated by reference.
After adding the carrier, the unit dosage
amounts (i.e., for IL-2 volumes that will provide 0.01
to 2 mg, preferably 0.2 to 1.0 mg, IL-2 per dose) of the
solution are dispensed into containers, the containers
are capped with a slotted stopper, and the contents are
lyophilized using conventional freeze-drying conditions
and apparatus.
The lyophilized, sterile product consists of a
mixture of (1) IL-2, (2) carrier (dextrose, sucrose, or
mannitol), (3) optionally other excipients such as human
serum albumin, Tween*80, and the like and (4) a small
amount of buffer that will provide a physiological pH
when the mixture is reconstituted. The product may also
contain a minor amount of a preservative to enhance
chemical stability. The recombinant IL-2 will typically
constitute about 0.015% to 10% by dry weight of the
mixture, more preferably about 2% to 5% of the mixture.
The lyophilized mixture may be reconstituted
by injecting a conventional parenteral aqueous injection
such as distilled water for injection, Ringer's solution
injection, Hank's solution injection, dextr~se injec-
tion, dextrose and salt injection, physiological salineinjection, or the like, into the vial. The injection
should be added against the side of the vial to avoid
excess foaming. The amount of injection added to the
vial will typically be in the range of 1 to 5 ml,
preferably 1 to 2 ml.
In an alternative formulation, described in
PCT W087/00056, published 15 January 1987, entitled
"Solubilization of Recombinant Proteins for Pharma-
ceutical Compositions Using Homopolymer Conjugation" to
M. Knauf et al,
*Trade mark
1337~71
-23-
The IL-2 is reacted ~Jith an
activated polymer selected from polyethylene glycol,
homopolymers and polyoxyethylated polyols such as
polyoxyethylated glycerol. The polymer preferably has a
molecular weight of from 300 to 100,000 daltons, more
preferably 350 to 40,000 daltons. The polymer is
activated by conjugation with a coupling agent having
terminal groups reactive with both the free amine or
thiol groups of the protein and the hydroxyl group of
the polymer. Examples of such coupling agents include
hydroxynitrobenzene sulfonic ester, cyanuric acid
chloride, and N-hydroxysuccinimide. The IL-2 is then
formulated directly with the water-soluble carrier and
buffer as described above, the formulation is
lyophilized, and the lyophilized mixture may be
reconstituted as described above.
The reconstituted formulation prepared as
described above is suitable for parenteral and oral
administration to humans or other mammals in
therapeutically effective amounts (i.e., amounts which
eliminate or reduce the patient's pathological
condition) to provide therapy thereto. IL-2 therapy is
appropriate for a variety of immunomodulatory~
indications such as T cell mutagenesis, induction of
cytotoxic T cells, augmentation of natural killer cell
activity, induction of IFN-y, restoration and
enhancement of cellular immunity (e.g., treatment of
immune deficient conditions), and augmentation of cell-
mediated anti-tumor activity.
The formulations of this invention are useful
for parenteral administration, for example, intravenous,
subcutaneous, intramuscular, intraorbital, ophthalmic,
intracapsular, intraspinal, intrasternal, topical,
intranasal aerosol, scarification, and also, for oral
1337671
-24-
administration. The preferred routes of administration
are by intramuscular, subcutaneous and intravenous
injection, and by topical administration. The use of
nonionic detergents are especially preferred for
topically administered formulations because of their
ability to penetrate the skin surface.
The following examples further illustrate the
process and composition of the invention. These
examples are not intended to limit the invention in any
manner. In these examples all temperatures are in
degrees Celsius unless otherwise indicated. Figure 1
indicates the preferred process of this invention,
represented by the examples.
Example 1
This example illustrates a preferred process
for recovering, purifying and formulating recombinant
IL-2.
Des-alanyl-IL-2serl2s was recovered from E.
coli. The strain of des-alanyl-IL-2serl25-producing E-
coli (K12/MM294-1) carrying plasmid pLW45 used in this
example was deposited at the American Type Culture
Collection of 4 March 1984 under accession number
39,626. Said analog and a method of preparation are
disclosed in U.S. Patent No. 4,518,584.
The E. coli thus transformed with plasmid
pLW45 were grown in a 1000-liter fermenter at 37C. The
dissolved oxygen was maintained at about 40% by, as
necessary, (1) increasing agitation; (2) adding air; and
(3) adding oxygen.
Once the fermenter was filled with water to
the operating volume, the following trace elements were
added:
1 33767~
-25-
-
ZnS04 7H20 30 ~M
MnS04 4H20 30 ~M
CuS04 5H20 3 ~M
Na3 citrate 2H20 1.5 mM
KH2Po4 21 mM
(NH4)2so4 72 mM.
The fermenter feed and addition vessels were then
sterilized according to standard operating procedures.
Then the following sterile additions were made:
MgS04 7H20 3 mM
FeS04 7H20 72 ~M
L-tryptophan 70 mg/L
thiamine HCl 20 mg/L
glucose 50 g/L
tetracycline 5 mg/L.
The fermenter was cooled and inoculated with frozen or
seed E. coli culture at 2 mg/L dry weight cells.
Throughout .fermentation, the pH is maintained at 6.8
using KOH. Optical density measurements and residual
glucose measurements on samples were taken~ at 14-16
hours and approximately one hour intervals thereafter.
Induction of des-alanyl-IL-2serl25 production
by depletion of L-tryptophan from the culture medium
occurred at about OD6go=10 followed by the addition of
casamino acids to a final concentration of 2% at
OD680=15- Cultures were harvested about 3-5 hours
later.
The refractile bodies containing the des-
alanyl-IL-2-serl2s were then isolated. The harvested
material was concentrated about 5-10 fold by circulating
1 337671
-26-
the harvest material under pressure through UF cross-
flow filtration cartridges with a 100 K molecular weight
cutoff. The cells were disrupted by 3 passes through a
disrupter at about 6500 psi (195 atm). EDTA was then
added to a final concentration of 5 mM. The suspension
was diafiltered against 5 volumes of deionized water.
Octanol was added to 1% (v/w) to kill any residual live
bacteria in the diafiltered product. 2 mM EDTA was
added and after several hours, the diafiltered disrup-
tate was redisrupted by passing it through a disrupter.
Sucrose was added to the redisruptate to
create a final density between 1.1 and 1.25 g/ml. The
mixture was centrifuged at 8,000 to 20,000 x g at 1-2
lpm, and the particle pellet or paste was collected. A
temperature of at least 20C was maintained prior to and
during centrifugation.
The particle paste was then mixed with 17 ml
per gram of paste of an aqueous solution of saturated
guanidine hydrochloride, DTT, 50 mM, Tris, 50 mM, and
25 mM EDTA, the pH was adjusted to 8.0 with NaOH and
heated to 40C for about 10 min. Undissolved materials
were removed from the mixture by centrifugation at
3000 x g for 15 min.
The next step in the purification was to
remove the DTT and EDTA from the IL-2 solution
(supernatant) by gel filtration using a Sephadexr~ G-25
column. The column was run in 7 M guanidine
hydrochloride buffer at pH 7.5. Using a process
chromatogram, the IL-2 peak was collected and the peak
was diluted with guanidine hydrochloride buffer to a
protein concentration of 0.5 mg/ml.
Oxidation of th~e IL-2 was initiated by adding
CuC12 in a molar ratio of 3:1 (CuC12 to IL-2). The
oxidation was carried out at about 25C in 7 M guanidine
1337671
-27-
hydrochloride, 10 mM phosphate. The pH was controlled
at 7.5 + 0.2 during oxidation and 4 mM EDTA was added
when the oxidation was completed. Since oxidized IL-2
is more hydrophilic than reduced IL-2, the progress of
the oxidation reaction was monitored by RP-HPLC.
The resulting solution of oxidized IL-2 was
then diluted with 10 mM phosphate buffer to reduce the
guanidine hydrochloride concentration to 2 M. The IL-2
concentration was then increased to 2.5 mg/ml using a
hollow fiber membrane ultrafiltration unit with a 10,000
dalton cutoff. The solution was then further diluted
with 10 mM phosphate buffer to 0.2 M guanidine
hydrochloride and allowed to sit overnight at 4C to
obtain a precipitate.
The precipitate consisting of extraneous E.
coli proteins and some IL-2 was then removed by
filtering with a cellulose acetate filter to obtain
approximately 85% recovery of refolded IL-2. (NH4)2SO4
was then added to the supernatant to a concentration of
1.25 M. This solution was loaded onto a Pharmacia
Phenyl Fast-Flow Sepharose hydrophobic interaction
column. IL-2 was recovered from the column with a
decreasing (NH4)2SO4 gradient with the IL-2 collected in
the fractions at about 0.95 to 0.75 M (NH4)2SO4. The
2 pooled fractions were diafiltered and then loaded on a
Pharmacia carboxymethyl (CM) Fast-Flow Sepharose ion
exchange column at pH 7 equilibrated with 10 mM
phosphate buffer. IL-2 fractions were recovered at
about 0.15 M NaCl.
The resulting IL-2 was 98~ pure by SDS-PAGE
analysis and homogeneous by HPLC analysis. Its specific
activity was 8 x 106 uni~ts/mg as measured by the HT-2
cell proliferation assay. Enzyme-linked immunosorbent
assays (ELISA) were carried out to determine whether
... ~,,
-28- 133767~
this renatured recombinant IL-2 (guanidine-process IL-2)
binds to polyclonal antibodies that bind to the
recombinant IL-2 made by the process described in U.S.
Patent No. 4,569,790, in which the IL-2 is solubilized
with SDS (SDS-process IL-2). Serum from a patient
treated with SDS-process IL-2 was diluted 1:1000 in
assay buffer (PBS with 0.5% BSA and 0.05% Tween 20) and
mixed with SDS or guanidine-process IL-2 in final
concentrations of 0 to 5 ~g/ml. After a two hour room
temperature incubation, the mixtures were applied in 100
~1 volumes to 96 well microelisa plates (Immulon I,
Dynatech) previously coated with SDS-process IL-2 (5
~g/ml in 0.05 M Na2CO3, pH 9.6, 100 ~l/well). The
mixtures were allowed to sit in the IL-2-coated wells
for thirty minutes, at which time the plates were
thoroughly washed in PBS with 0.05% Tween 20, and
peroxidase-conjugated goat anti-human IgG (Cappel,
1:1000 dilution) was added. After another two hour
incubation the second antibody was removed and substrate
added (OPD, Sigma, 100 ~l/well). The enzymatic reaction
was quenched twenty minutes later by the addition of 50
~1 2N HCl to each well. Absorbances at 490 nm were
measured using a Dynatech MR580 plate reader (reference
wavelength 405 nm). Plots of the absorbance versus the
amount of competing antigen are shown in Figure 2. As
shown the guanidine-process IL-2 did not compete
significantly.
Example 2
Example 1 was repeated through the IL-2
refolding step (recovery of supernatant following
reduction of guanidine hydrochloride to 0.2 M).
*Trade mark
1337~71
-29-
The IL-2 solution (8.8 mg of protein) was
acidified with trifluoroacetic acid to a pH of 2.1 and
then centrifuged to remove any precipitate. This was
loaded on a 1.25 cm by 30 cm column of Vydac C-4 silica
equilibrated with 0.1% trifluoroacetic acid in water.
The column was eluted with a gradient of acetonitrile
containing 0.1% trifluoroacetic acid. The fractions of
pure IL-2 were pooled and then dialyzed into 7 M
guanidine pH 7.5 buffer. This was then dialyzed against
mM phosphate buffer pH 7Ø A precipitate was
removed by microcentrifugation to recover 68% of the
IL-2 in the supernatant.
Example 3
Example 1 was repeated through the recovery of
the refractile body particle paste.
About 113.7 g of solid guanidine (7 M final
concentration) was added to ~14 g of the particle paste
followed by addition of 10 mM Tris/2 mM EDTA buffer to
about 190 ml. After homogenizing, -1.5 g of solid DTT
(50 mM final concentration) was added and the pH was
adjusted to 8-8.5 with NaOH. The solution was warmed to
50C for 15 mins to promote reduction. The solution was
then diluted with 10 mM Tris/2 mM EDTA to a final volume
of 200 ml.
The next step in the purification was to
remove the reducing agent and other contaminants from
the IL-2 material. About 25 ml of the reduced IL-2 were
diluted to approximately 5-10 mg IL-2/ml with 75 ml 7.0
M guanidine. This solution was diluted to 4.8 M
guanidine with Tris/EDTA buffer and allowed to stand two
hours. Very little precipitate was formed, and the
precipitate was removed by centrifugation at 10,000 x g
for 15 minutes. The supernatant was diluted to 4.0 M
1337 671
-30-
guanidine with Tris/EDTA buffer and allowed to stand two
hours at room temperature. The heavy precipitate was
collected by centrifugation (10,000 g x 15 min).
The pellet was washed once with 50 ml 2% Tween
80 and twice with 50 ml H2O. 16.7 g of solid guanidine
was added and the solution was brought to 25 ml with
water. The solubilized IL-2 was diluted to 1 mg/ml with
7.0 M guanidine in 10 mM Citrate, pH 6.5. CuC12 (to 0.1
mM) was added and the pH was adjusted to 8-8.5. This
solution was allowed to stir overnight.
Using a YM spiral-wound cartridge, the guani-
dine was diafiltered away and the IL-2 was concentrated
to about 2 mg/ml. Care was taken to remove air from the
system because soluble IL-2 is sensitive to agitation,
especially with air bubbles. It was diafiltered against
10 volumes of 2.5% sucrose and 140 mM NaCl in 10 mM
NaCitrate pH 6.5 solution. After centrifugation at 3000
g x 15 min, 600 ml of supernatant containing 0.63 mg/ml
of IL-2 was collected. The ionic strength was reduced
by dialysis into 10 mM NaCitrate, pH 6.5 before pro-
ceeding.
The chromatography consisted of two columns,
each equilibrated with 10 mM NaCitrate pH 6-.5 buffer.
The first column was packed with DEAE Sepharose Fast
Flow (Pharmacia). 25 ml of 0.63 mg IL-2/ml in 10 mM
NaCitrate pH 6.5 was run through a 1 x 10 cm column at
0.5 ml/minute. The pool's NaCl concentration was
adjusted to 40 mM NaCl. The second column was packed
with CM Sepharose Fast Flow (Pharmacia). 28.5 ml of
0.38 mg IL-2/ml was loaded at 0.5 ml/min. The IL-2
bound to the gel and was eluted with an increasing NaCl
gradient (40-400 mM NaCl ~in 10 mM NaCitrate pH 6.5, 0.3
ml/min) over 6 hours. About 12.6 ml of 0.71 mg of IL-2
per ml was pooled.
~ 337~71
-31-
The CM pool was desalted into lO mM sodium
citrate pH 6.5 over G25 Sephadex to remove NaCl and
provide a well characterized buffer for formulation.
The resulting IL-2 was over 99% pure by
analytical RP-HPLC.
Example 4
About 5 ml of IL-2-containing refractile
bodies, slurried with an equal volume of water was
dissolved with guanidine buffer. The solution had a
guanidine concentration of 7 M and a volume of 35 ml.
The pH of the solution was adjusted to about 8.0 with 3
M Tris base. Next, 0.3 9 of DTT was added and the
solution was heated to about 45C for 15 min. The
solution was then diluted with an equal volume of 0.1 M
citrate pH 5.0 buffer and allowed to stand for 1.5
hours. The formed precipitate was separated by
centrifugation (10,000 x 9 for 10-20 min). The
precipitate was washed four times with 70 ml of 3.5 M
guanidine and twice with 70 ml of water. The precipi-
tate was dissolved in 7 M guanidine and was analyzed by
reverse phase HPLC. Approximately 265 mg of IL-2 were
recovered in about 90% purity.
From the foregoing it may be seen that the
present process provides advantages as regards: (1)
simplicity of the purification process, (2) the absence
of solubilizing agent in the final product and (3)
recombinant IL-2 product that appears to be less
immunogenic than that previously made.
In addition to the aforedescribed vector
system employing the trp~promoter for IL-2 expression,
alternative vector systems include the use of the lambda
pL promoter and/or a positive retroregulatory element.
,~
-32- 1 337G71
These vector systems are described in U.S. Patent Nos.
4,711,845, issued 8 December 1987 and 4,666,848, issued
19 May 1987
Vector systems described in the aforedescribed
patents, as well as additional vectors provided below,
have been deposited with the American Type Culture
Collection (ATCC), 12301 Parklawn Drive, Rockville,
Maryland under the terms of the Budapest Treaty on the
International Recognition of the Deposit of Micro-
organisms for the Purpose of Patent Procedure andRegulations thereunder and are thus maintained and made
available according to the terms of the Budapest Treaty.
Availability of such strains is not to be construed as a
license to practice the invention in contravention of
the rights granted under the authority of any government
in accordance with its patent laws.
The deposited plasmids have been assigned the
indicated ATCC deposit numbers:
Plasmid ATCC No.Deposit Date
pFcss in E. coli DG95 l~a
(N7N53cI8575USP80) 398314 September 1984
pEC54.t in E. coli
DG95 lam~da 3978931 July 1984
pHCW701 in E. coli K12 MM29g 39757 8 June 198~
pLWl in E. coli K12 MM29g 39so525 July 1983
pLWg6 in E. coli K12 MM294 3945229 September 1983
pLW55 in E. coli K12 MM29~.1 39516 29 September 1983
pSY3001 in E. coli K12 MM294 39949 19 December 198g
Modifications of the above-described
modes for carrying out the invention which are obvious
to those of skill in the fields of biochemistry and
1337671
-33-
related fields are intended to be within the scope of
the following claims.
