U.S. patent application number 10/635865 was filed with the patent office on 2004-02-12 for activated protein c formulations.
Invention is credited to Carlson, Andrew David, Sheliga, Theodore Arsay.
Application Number | 20040028670 10/635865 |
Document ID | / |
Family ID | 46203838 |
Filed Date | 2004-02-12 |
United States Patent
Application |
20040028670 |
Kind Code |
A1 |
Carlson, Andrew David ; et
al. |
February 12, 2004 |
Activated protein C formulations
Abstract
The present invention relates to pharmaceutical formulations of
activated protein C. The activated protein C formulations of the
present invention are more stable than other formulations of
activated protein C and demonstrate fewer degradation products over
time.
Inventors: |
Carlson, Andrew David;
(Indianapolis, IN) ; Sheliga, Theodore Arsay;
(Indianapolis, IN) |
Correspondence
Address: |
ELI LILLY AND COMPANY
PATENT DIVISION
P.O. BOX 6288
INDIANAPOLIS
IN
46206-6288
US
|
Family ID: |
46203838 |
Appl. No.: |
10/635865 |
Filed: |
August 6, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10635865 |
Aug 6, 2003 |
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09545175 |
Apr 6, 2000 |
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6630137 |
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09545175 |
Apr 6, 2000 |
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09065975 |
Apr 24, 1998 |
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6159468 |
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60045255 |
Apr 28, 1997 |
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Current U.S.
Class: |
424/94.1 ;
514/53 |
Current CPC
Class: |
C12Y 304/21069 20130101;
C12N 9/6464 20130101; A61K 38/4866 20130101 |
Class at
Publication: |
424/94.1 ;
514/53 |
International
Class: |
A61K 038/43; A61K
031/7012 |
Claims
We claim:
1. A lyophilized formulation consisting essentially of activated
protein C; a salt; a bulking agent selected from mannitol,
trehalose, raffinose, and sucrose, and mixtures thereof; and a
buffer system such that upon reconstitution the resulting
formulation has a pH between about 5.5 and about 6.1.
2. The formulation of claim 1, wherein the salt is potassium
chloride or sodium chloride and the buffer system is selected from
Tris-acetate, sodium citrate, and sodium phosphate, or mixtures
thereof.
3. The formulation of claim 2, wherein the resulting formulation
has a pH between about 5.9 and about 6.1.
4. The formulation of claim 2, wherein the resulting formulation
has a pH between about 5.6 and about 6.0.
5. The formulation of claim 2, wherein the Resulting formulation
has a pH between about 5.8 and about 6.1.
6. The formulation of claim 2, wherein the resulting formulation
has a pH of about 6.0.
7. A lyophilized formulation consisting of activated protein C; a
salt; a bulking agent selected from mannitol, trehalose, raffinose,
and sucrose, and mixtures thereof; and a buffer system such that
upon reconstitution the resulting formulation has a pH between
about 5.5 and about 6.0.
8. The formulation of claim 7, wherein the salt is sodium chloride
and the buffer system is selected from Tris-acetate, sodium
citrate, and sodium phosphate, or mixtures thereof.
9. The formulation of claim 8, wherein the resulting formulation
has a pH between about 5.9 and about 6.1.
10. The formulation of claim 8, wherein the resulting formulation
has a pH between about 5.6 and about 6.0.
11. The formulation of claim 8, wherein the resulting formulation
has a pH between about 5.8 and about 6.1.
12. The formulation of claim 8, wherein the buffer is sodium
citrate and the resulting formulation has a pH of about 6.0.
Description
[0001] This application is a continuation-in-part application of
co-pending U.S. patent application Ser. No. 09/065,975 filed Apr.
24, 1998, which claimed the benefit of U.S. Provisional Application
No. 60/045,255, filed Apr. 28, 1997.
FIELD OF THE INVENTION
[0002] This invention is in the field of human medicine,
particularly in the treatment of vascular disorders with activated
protein C. More specifically, the present invention relates to
formulations of activated human protein C.
BACKGROUND OF THE INVENTION
[0003] Protein C is a serine protease and naturally occurring
anticoagulant that plays a role in the regulation of homeostasis by
inactivating Factors V.sub.a and VIII.sub.a in the coagulation
cascade. Human protein C is made in vivo primarily in the liver as
a single polypeptide of 461 amino acids. This single chain
precursor molecule undergoes multiple post-translational
modifications including 1) cleavage of a 42 amino acid signal
sequence; 2) proteolytic removal from the one chain zymogen of the
lysine residue at position 156 and the arginine residue at position
157 to make a 2-chain zymogen form of the molecule, (i.e., a light
chain of 155 amino acid residues attached through a disulfide
bridge to the serine protease-containing heavy chain of 262 amino
acid residues); 3) vitamin K-dependent carboxylation of nine
glutamic acid residues clustered in the first 42. amino acids of
the light chain, resulting in nine gamma-carboxyglutamic acid
residues; and 4) carbohydrate attachment at four sites (one in the
light chain and three in the heavy chain). The heavy chain contains
the well established serine protease triad of Asp 257, His 211 and
Ser 360. Finally, the circulating 2-chain zymogen is activated in
vivo by thrombin at a phospholipid surface in the presence of
calcium ion. Activation results from removal of a dodecapeptide at
the N-terminus of the heavy chain, producing activated protein C
(aPC) possessing enzymatic activity.
[0004] In addition to the enzymatic activities of aPC within the
blood coagulation cascade, aPC also can autodegrade, leading to
decreased functionality as an anticoagulant. Applicants have
discovered an important degradation pathway. Autodegradation of the
N-terminus of the light chain may result in a clip on either side
of the histidine residue at position 10. Thus, this degradation
pathway yields two inactive products: 1) des(1-9) activated protein
C, wherein the first nine N-terminal residues of the light chain
have been removed; and 2) des(1-10) activated protein C, wherein
the first ten N-terminal residues of the light chain have been
removed. This degradation pathway, which has not been previously
reported, results in loss of anticoagulant activity due to the
removal of the critical GLA residues at positions 6 and 7.
Therefore, minimizing the level of the des(1-9) and des(1-10)
activated Protein C autodegradation products is important in
achieving a potent, high purity, activated protein C pharmaceutical
formulation. These variants were previously unknown degradation
products and are exceedingly difficult, if not impossible, to
remove by conventional purification techniques. Applicants have
further discovered that solid-state solubility is significantly
enhanced in the presence of a select group of bulking agents.
[0005] It is clearly desirable to minimize such degradation of
activated protein C in both the solution and lyophilized. solid
states. Accordingly, these discoveries allow the preparation of
potent, high purity, activated protein C formulations which are
pharmaceutically elegant to the health care provider.
[0006] The present invention provides improved formulations of
activated protein C substantially free of such autodegradation
products, particularly, des(1-9) and des(1-10) forms of the light
chain of activated protein C. Therefore, said formulations are
suitable for administration to a patient in need thereof.
SUMMARY OF THE INVENTION
[0007] The present invention provides a stable lyophilized
formulation which may comprise, consist of, or consist essentially
of activated protein C and a bulking agent selected from the group
consisting of mannitol, trehalose, raffinose, sucrose, and mixtures
thereof.
[0008] An aspect of the invention provides a lyophilized
formulation consisting essentially of activated protein C; a salt;
a bulking agent selected from mannitol, trehalose, raffinose, and
sucrose, and mixtures thereof; and a buffer system such that upon
reconstitution the resulting formulation has a pH between about 5.5
and about 6.1.
[0009] A preferred aspect of the invention provides a lyophilized
formulation consisting of activated protein C; a salt; a bulking
agent selected from mannitol, trehalose, raffinose, and sucrose,
and mixtures thereof; and a buffer system such that upon
reconstitution the resulting formulation has a pH between about 5.5
and about 6.0.
[0010] The present invention also provides a stable lyophilized
formulation comprising, consisting of, or consisting essentially of
about 2.5 mg/mL activated protein C, about 15 mg/mL sucrose, and
about 20 mg/mL NaCl. Furthermore, the present invention provides a
stable lyophilized formulation comprising, consisting of, or
consisting essentially of about 5 mg/mL activated protein C, about
30 mg/mL sucrose, and about 38 mg/mL NaCl.
[0011] The present invention also provides a process for preparing
a formulation comprising activated protein. C and a bulking agent
selected from the group consisting of mannitol, trehalose,
raffinose, and sucrose and mixtures thereof.
[0012] The invention also provides a unit dosage form comprising,
consisting of, or consisting essentially of a unit dosage
receptacle containing the formulation wherein the weight to weight
ratio is about 1 part activated protein C, about 7.6 parts salt and
about 6 parts bulking agent.
[0013] The invention further provides a method of treating disease
states involving intravascular coagulation comprising the
administration of a formulation of activated protein C described
herein.
DETAILED DESCRIPTION OF THE INVENTION
[0014] For purposes of the present invention, as disclosed and
claimed herein, the following terms are as defined below.
[0015] aPC or activated protein C refers to activated protein C
whether recombinant or plasma derived. aPC includes and is
preferably human activated protein C although aPC may also include
other species or derivatives having protein C proteolytic,
amidolytic, esterolytic, and biological (anticoagulant or
pro-fibrinolytic) activities. Examples of protein C derivatives are
described by Gerlitz, et al., U.S. Pat. No. 5,453,373, and Foster,
et al., U.S. Pat. No. 5,516,650, the entire teachings of which are
hereby incorporated by reference.
[0016] APTT--activated partial thromboplastin time.
[0017] r-hPC--recombinant human protein C zymogen.
[0018] r-aPC--recombinant activated protein C produced by
activating protein C zymogen in vitro or in vivo or by direct
secretion of the activated form of protein C from procaryotic
cells, eukaryotic cells, or transgenic animals including, for
example, secretion from human kidney 293 cells as a zymogen then
purified and activated by techniques well known to the skilled
artisan and demonstrated in Yan, U.S. Pat. No. 4,981,952, and
Cottingham, WO 97/20043, the entire teachings of which are herein
incorporated by reference.
[0019] Continuous infusion--continuing substantially uninterrupted
the introduction of a solution into a blood vessel for a specified
period of time.
[0020] Bolus injection--the injection of a drug in a defined
quantity (called a bolus) at once.
[0021] Suitable for administration--a lyophilized formulation or
solution that is appropriate to be given as a therapeutic
agent.
[0022] Zymogen--protein C zymogen, as used herein, refers to
secreted, inactive forms, whether one chain or two chains, of
protein C.
[0023] Pharmaceutically acceptable buffer--a pharmaceutically
acceptable buffer is known in the art. Pharmaceutically acceptable
buffers include sodium phosphate, sodium citrate, sodium acetate,
or TRIS.
[0024] Activated protein C is an antithrombotic agent with a wider
therapeutic index than available anticoagulants, such as heparin
and the oral hydroxycoumarin type anticoagulants. As an
antithrombotic agent, aPC has a profound effect on the treatment of
a wide variety of acquired disease states involving intravascular
coagulation, including thrombotic stroke, deep vein thrombosis,
pulmonary embolism, peripheral arterial thrombosis, emboli
originating from the heart or peripheral arteries, acute myocardial
infarction, disseminated intravascular coagulation, and acute pre
or postcapillary occlusions, including transplantations or retina
thrombosis.
[0025] The present invention relates to formulations of activated
protein C. The desired formulation would be one that is a stable
lyophilized product of high purity consisting of activated protein
C and a bulking agent selected from the group consisting of
mannitol, trehalose, raffinose, and sucrose. The lyophilized
product is reconstituted with the appropriate diluent such as
sterile water or sterile saline. Preferably, the resulting solution
has a pH of about 5.5 to about 6.5. Additional preferred pH ranges
include a pH range between about 5.5 and about pH 6.0; a pH between
about 5.5 and about pH 6.1; a pH between about 5.5 and about pH
6.2; a pH between about 5.6 and about pH 6.0; a pH between about
5.6 and about pH 6.1; a pH between about 5.6 and about pH 6.2; a pH
between about 5.7 and about pH 6.0; a pH between about 5.7 and
about pH 6.1; a pH between about 5.8 and about pH 6.0; and a pH
between about 5.8 and about pH 6.1.
[0026] The molecular interactions in a formulation between
activated protein C, buffer, salt concentration, pH, temperature,
and bulking agents, are complex, and the role that each factor
contributes to the stability of the formulation is unpredictable.
For example, controlling the pH, ionic strength, and preferably
temperature, the autodegradation of activated protein C in solution
during processing and in formulations can be reduced to levels
previously unobtainable--particularly in the absence of urea or
other denaturing agents, histidine, lysine hydrochloride, or
albumin. The lyophilized formulations of the present invention
provide stable, enzymatically active, activated protein C upon
resuspension because of reduced autodegradation. The present
invention has particularly reduced levels of des(1-9) aPC and
des(1-10) aPC. Generally, the levels of des(1-9) and des(1-10) aPC
are less than 10% of the autodegradation product. Preferably, the
levels of des(1-9) and des(1-10) aPC are less than 8% of the
autodegradation product. Still more preferably, the levels of
des(1-9) and des(1-10) aPC are less than 5% and most preferably
less than 3% of the autodegradation product. This stability is
obtained through careful control of the processing conditions and
by the addition of sucrose, trehalose, raffinose, or mannitol.
Interestingly, other bulking agents such as hydroxyethyl starch and
glycine do not offer the necessary stability or pharmaceutical
elegance.
[0027] The bulking agents of the present invention provide a
pharmaceutically elegant formulation which has a uniform appearance
and is readily solubilized when resuspended with the appropriate
solute. Upon reconstitution, the formulation is stable for up to 24
hours to 48 hours at room temperature. Resulting in stability
previously unachievable.
[0028] Preferred bulking agents in the formulation of activated
protein C are sucrose, trehalose and raffinose. More preferred
bulking agents are sucrose and raffinose and the most preferred
bulking agent is sucrose. The amount of bulking agent in the
formulation is 1 part aPC to 1 to 10 parts bulking agent on a
weight to weight basis. Moreover, the bulking agent concentration
of the formulation is an important formulation variable of the
freeze drying process. The optimum concentration of bulking agent
is dependent on the amount of aPC and species of bulking agent
selected. The preferred concentration of sucrose in the freezing
solution is 10 to 40 mg/mL. A more preferred concentration of
sucrose is 15 to 30 mg/mL. The most preferred concentration of
sucrose in the freezing solution is 15 mg/mL in a formulation of
aPC at 2.5 mg/mL. The most preferred concentration of sucrose in
the freezing solution is 30 mg/mL in a formulation of aPC at 5.0
mg/mL. The presence of the claimed bulking agent in the formulation
of activated protein C offers increased chemical and physical
stability.
[0029] Prior to freeze drying and upon reconstitution, it is
preferable to maintain the pH in the range of 5.5 to 6.5 to
minimize solution state autodegradation. The preferred pH of the
formulation is a pH between about pH 5.6 and about pH 6.4. More
preferred is a pH between about 5.7 to about 6.3. Even more
preferred is a pH between about 5.8 to about 6.2. Still even more
preferred is a pH between about 5.9 to about 6.1. The most
preferred pH is about pH 6.0. Additional preferred pH ranges
include a pH range between about 5.5 and about pH 6.0; a pH between
about 5.5 and about pH 6.1; a pH between about 5.5 and about pH
6.2; a pH between about 5.6 and about pH 6.0; a pH between about
5.6 and about pH 6.1; a pH between about 5.6 and about pH 6.2; a pH
between about 5.7 and about pH 6.0; a pH between about 5.7 and
about pH 6.1; a pH between about 5.8 and about pH 6.0; and a pH
between about 5.8 and about pH 6.1.
[0030] To maintain effective pH control, the aPC solution should
contain a pharmaceutically acceptable buffer. Accordingly, upon
freeze-drying, the formulation optionally and preferably comprises
a pharmaceutically acceptable buffer. Representative buffer systems
include Tris-acetate, sodium citrate, and sodium phosphate. More
preferred buffer systems include sodium citrate and sodium
phosphate. The most preferred buffer is sodium citrate. The
preferred molarity of the buffer system is 10 mM to 50 mM. A more
preferred molarity of the buffer system is 10 mM to 20 mM. The most
preferred molarity is 40 mM. The skilled artisan will recognize
that many other buffer systems are available which also can be used
in the formulations of the present invention.
[0031] Similarly, during freeze drying and upon reconstitution, the
ionic strength is a critical variable to ensure solution state
stability. The ionic strength is generally determined by the salt
concentration of the solution. Pharmaceutically acceptable salts
typically used to generate ionic strength include but are not
limited to potassium chloride (KCl) and sodium chloride (NaCl). The
preferred salt in the present invention is sodium chloride. During
freeze-drying, the salt concentration must be high enough to cause
the salt to crystallize during the freezing step of the
freeze-drying cycle. Preferably, the sodium chloride concentration
is greater than 150 mM. More preferably, the sodium chloride
concentration in the freezing solution is between 150 mM to 1000
mM. For a formulation containing 2.5 mg/mL aPC, the more preferable
sodium chloride concentration in the freezing solution is between
150 mM to 650 mM. Even more preferably the sodium chloride
concentration in the freezing solution is between 250 mM to 450 mM.
Still even more preferably the sodium chloride concentration in the
freezing solution is between 300 mM to 400 mM. The most preferable
sodium chloride concentration in the freezing solution is 325 mM
for a formulation containing 2.5 mg/mL aPC.
[0032] Similarly, for a formulation containing 5.0 mg/mL aPC, the
more preferable sodium chloride concentration in the freezing
solution is between 150 mM to 1000 mM. Even more preferably the
sodium chloride concentration in the freezing solution is between
250 mM to 750 mM. Still even more preferably the sodium chloride
concentration in the freezing solution is between 400 mM to 700 mM.
The most preferable sodium chloride concentration in the freezing
solution is 650 mM for a formulation containing 5.0 mg/mL aPC.
[0033] The ratio of aPC:salt:bulking agent (w:w:w) is an important
factor in a formulation suitable for the freeze drying process. The
ratio varies depending on the concentration of aPC, salt selection
and concentration and bulking agent selection and concentration.
One skilled in the art could readily identify the preferred ratio
of aPC:salt:bulking agent by techniques appreciated in the art and
described, for example, in Example 1. Particularly, a weight ratio
of one part activated protein C to between about 7 to 8 parts salt
to between about 5 to 7 parts bulking agent is preferred. More
preferred is a weight ratio of one part activated protein C to
between about 7.5 to about 8 parts salt to between about 5.5 to
about 6.5 parts bulking agent. Most preferred is a ratio of about 1
part activated protein C to about 7.6 parts salt to about 6 parts
bulking agent.
[0034] The preferred salt is sodium chloride at a concentration of
325 mM (for a formulation containing 2.5 mg/mL aPC) and 650 mM (for
a formulation containing 5.0 mg/mL aPC) and at a ratio of about
1.3:1 with sucrose (w:w). This concentration is high enough to
cause the salt to crystallize during the freezing process, most
likely resulting in an amorphous mixture of aPC, sucrose, and
citrate that can be lyophilized. Thus, the ionic strength of NaCl
at the preferred concentrations of 325 mM and 650 mm convey a
stability to the formulation during the freeze-drying process.
[0035] The present invention further provides a process for
preparing a stable lyophilized formulation which comprises
lyophilizing a solution comprising activated protein C and a
bulking agent selected from the group consisting of mannitol,
trehalose, raffinose, and sucrose, and mixtures thereof. The
invention also provides a process for preparing a stable
lyophilized formulation which comprises, consists essentially of or
consists of, lyophilizing a solution comprising, consists
essentially of or consists of, about 2.5 mg/mL activated protein C,
about 15 mg/mL sucrose, about 19 mg/mL NaCl, and a sodium citrate
buffer having a pH greater than 5.5 but less than 6.5. Furthermore,
the present invention provides a process for preparing a stable
lyophilized formulation which comprises, consists eventually of or
consists of, lyophilizing a solution comprising about 5 mg/mL
activated protein C, about 30 mg/mL sucrose, about 38 mg/mL NaCl,
and a citrate buffer having a pH greater than 5.5 but less than
6.5.
[0036] The present invention provides a unit dosage form comprising
a unit dosage receptacle containing a stable lyophilized
formulation comprising activated protein C and a bulking agent
selected from the group consisting of mannitol, trehalose,
raffinose, and sucrose, and mixtures thereof. Furthermore, the
present invention provides a method of treating disease states
involving intravascular coagulation comprising the administration
of said formulation.
[0037] The aPC is preferably administered parenterally to ensure
its delivery into the bloodstream in an effective form by injecting
the appropriate dose as continuous infusion for about one to about
forty-eight hours. The amount of aPC administered is from about
0.01 mg/kg/hr to about 0.05 mg/kg/hr. The continuous infusion is
preferably administered at about 0.024 mg/kg/hr for 96 to 144
hours. Alternatively, the aPC will be administered by injecting a
portion of the appropriate dose per hour as a bolus injection over
a time from about 5 minutes to about 30 minutes, followed by
continuous infusion of the appropriate dose for about twenty-three
hours to about 47 hours which results in the appropriate dose
administered over 24 hours to 48 hours.
[0038] The following examples will help describe how the invention
is practiced and will illustrate the invention. The scope of the
present invention is not to be construed as merely consisting of
the following examples.
PREPARATION 1
Preparation of Human Protein C
[0039] Recombinant human protein C (r-hPC) was produced in Human
Kidney 293 cells by techniques well known to the skilled artisan
such as those set forth in Yan, U.S. Pat. No. 4,981,952, the entire
teaching of which is herein incorporated by reference. The gene
encoding human protein C is disclosed and claimed in Bang, et al.,
U.S. Pat. No. 4,775,624, the entire teaching of which is
incorporated herein by reference. The plasmid used to express human
protein C in 293 cells was plasmid pLPC which is disclosed in Bang,
et al., U.S. Pat. No. 4,992,373, the entire teaching of which is
incorporated herein by reference. The construction of plasmid pLPC
is also described in European Patent Publication No. 0 445 939, and
in Grinnell, et al., 1987, Bio/Technology 5:1189-1192, the
teachings of which are also incorporated herein by reference.
Briefly, the plasmid was transfected into 293 cells, then stable
transformants were identified, subcultured and grown in serum-free
media. After fermentation, cell-free medium was obtained by
microfiltration.
[0040] The human protein C was separated from the culture fluid by
an adaptation of the techniques of Yan, U.S. Pat. No. 4,981,952.
The clarified medium was made 4 mM in EDTA before it was absorbed
to an anion exchange resin (Fast-Flow Q, Pharmacia). After washing
with 4 column volumes of 20 mM Tris, 200 mM NaCl, pH 7.4 and 2
column volumes of 20 mM Tris, 150 mM NaCl, pH 7.4, the bound
recombinant human protein C zymogen was eluted with 20 mM Tris, 150
mM NaCl, 10 mM CaCl.sub.2, pH 7.4. The eluted protein was greater
than 95% pure after elution as judged by SDS-polyacrylamide gel
electrophoresis.
[0041] Further purification of the protein was accomplished by
making the protein 3 M in NaCl followed by adsorption to a
hydrophobic interaction resin (Toyopearl Phenyl 650 M, TosoHaas)
equilibrated in 20 mM Tris, 3 M NaCl, 10 mM CaCl.sub.2, pH 7.4.
After washing with 2 column volumes of equilibration buffer without
CaCl.sub.2, the recombinant human protein C was eluted with 20 mM
Tris, pH 7.4.
[0042] The eluted protein was prepared for activation by removal of
residual calcium. The recombinant human protein C was passed over a
metal affinity column (Chelex-100, Bio-Rad) to remove calcium and
again bound to an anion exchanger (Fast Flow Q, Pharmacia). Both of
these columns were arranged in series and equilibrated in 20 mM
Tris, 150 mM NaCl, 5 mM EDTA, pH 7.4. Following loading of the
protein, the Chelex-100 column was washed with one column volume of
the same buffer before disconnecting it from the series. The anion
exchange column was washed with 3 column volumes of equilibration
buffer before eluting the protein with 0.4 M NaCl, 20 mM
Tris-acetate, pH 6.5. Protein concentrations of recombinant human
protein C and recombinant activated protein C solutions were
measured by UV 280 nm extinction E.sup.0.1%=1.81 or 1.85,
respectively.
PREPARATION 2
Activation of Recombinant Human Protein C
[0043] Bovine thrombin was coupled to Activated CH-Sepharose 4B
(Pharmacia) in the presence of 50 mM HEPES, pH 7.5 at 4.degree. C.
The coupling reaction was done on resin already packed into a
column using approximately 5000 units thrombin/mL resin. The
thrombin solution was circulated through the column for
approximately 3 hours before adding 2-aminoethanol (MEA) to a
concentration of 0.6 mL/L of circulating solution. The
MEA-containing solution was circulated for an additional 10-12
hours to assure complete blockage of the unreacted amines on the
resin. Following blocking, the thrombin-coupled resin was washed
with 10 column volumes of 1 M NaCl, 20 mM Tris, pH 6.5 to remove
all non-specifically bound protein, and was used in activation
reactions after equilibrating in activation buffer.
[0044] Purified r-hPC was made 5 mM in EDTA (to chelate any
residual calcium) and diluted to a concentration of 2 mg/mL with 20
mM Tris, pH 7.4 or 20 mM Tris-acetate, pH 6.5. This material was
passed through a thrombin column equilibrated at 37.degree. C. with
50 mM NaCl and either 20 mM Tris pH 7.4 or 20 nM Tris-acetate pH
6.5. The flow rate was adjusted to allow for approximately 20 min.
of contact time between the r-hPC and thrombin resin. The effluent
was collected and immediately assayed for amidolytic activity. If
the material did not have a specific activity (amidolytic)
comparable to an established standard of aPC, it was recycled over
the thrombin column to activate the r-hPC to completion. This was
followed by 1:1 dilution of the material with 20 mM buffer as
above, with a pH of either 7.4 or 6.5 to keep the aPC at lower
concentrations while it awaited the next processing step.
[0045] Removal of leached thrombin from the aPC material was
accomplished by binding the aPC to an anion exchange resin (Fast
Flow Q, Pharmacia) equilibrated in activation buffer (either 20 mM
Tris, pH 7.4 or 20 mM Tris-acetate, pH 6.5) with 150 mM NaCl.
Thrombin does not interact with the anion exchange resin under
these conditions, but passes through the column into the sample
application effluent. Once the aPC is loaded onto the column, a 2-6
column volume wash with 20 mM equilibration buffer is done before
eluting the bound aPC with a step elution using 0.4 M NaCl in
either 5 mM Tris-acetate, pH 6.5 or 20 mM Tris, pH 7.4. Higher
volume washes of the column facilitated more complete removal of
the dodecapeptide. The material eluted from this column was stored
either in a frozen solution (-20.degree. C.) or as a lyophilized
powder.
[0046] The anticoagulant activity of activated protein C was
determined by measuring the prolongation of the clotting time in
the activated partial thromboplastin time (APTT) clotting assay. A
standard curve was prepared in dilution buffer (1 mg/mL
radioimmunoassay grade bovine serum albumin [BSA], 20 mM Tris, pH
7.4, 150 mM NaCl, 0.02% NaN.sub.3) ranging in protein C
concentration from 125-1000 ng/mL, while samples were prepared at
several dilutions in this concentration range. To each sample
cuvette, 50 .mu.L of cold horse plasma and 50 .mu.L of
reconstituted activated partial thromboplastin time reagent (APTT
Reagent, Sigma) were added and incubated at 37.degree. C. for 5
min. After incubation, 50 .mu.L of the appropriate samples or
standards were added to each cuvette. Dilution buffer was used in
place of sample or standard to determine basal clotting time. The
timer of the fibrometer (CoA Screener Hemostasis Analyzer, American
Labor) was started immediately after the addition of 50 .mu.L
37.degree. C. 30 mM CaCl.sub.2 to each sample or standard.
Activated protein C concentration in samples are calculated from
the linear regression equation of the standard curve. Clotting
times reported here are the average of a minimum of three
replicates, including standard curve samples.
EXAMPLE 1
Formulation of Activated Protein C
[0047] The human activated protein C was prepared as described in
Preparations 1 and 2. The activated protein C formulations were
analyzed for processing in a conventional freeze dryer.
Freeze-Drying Microscopy and Differential Scanning Calorimetry
(DSC) were used to measure two parameters that determine if a
formulation can be processed in a conventional freeze dryer.
Freeze-Dry Microscopy is a useful technique in determining the
collapse temperatures of the frozen solutions that are to be
lyophilized. DSC is a useful technique in determining the
glass-transition temperature (Tg') of the frozen solution. The
collapse and glass-transition temperatures are especially helpful
in predicting the upper temperature limits that can be safely used
during the freeze-drying process. Results of Freeze-Drying
Microscopy are complimentary to the glass-transition temperature of
the Tg', values obtained by DSC. A collapse temperature above
-40.degree. C. is optimal for the sample to be processed in a
conventional freeze-dryer.
1TABLE 1 Freeze dry processing of aPC formulation matrices
Formulation Matrix aPC Sucrose NaCl Collapse Conc. Conc. Conc.
Temperature 2.5 mg/mL 15 mg/mL 50 mM -59.degree. C. 2.5 mg/mL 15
mg/mL 150 mM -60.degree. C. 2.5 mg/mL 15 mg/mL 325 mM -37.degree.
C. 5.0 mg/mL 30 mg/mL 50 mM -50.degree. C. to -45.degree. C. 5.0
mg/mL 30 mg/mL 150 mM -60.degree. C. to -55.degree. C. 5.0 mg/mL 30
mg/mL 325 mM -64.degree. C. 5.0 mg/mL 30 mg/mL 650 mM -32.degree.
C. to -28.degree. C.
[0048] The ratio of aPC to sucrose to sodium chloride (in 10 or 20
mM citrate buffer) is an important formulation variable affecting
the collapse and glass-transition temperatures. To be processed in
a conventional freeze-dryer, the sodium chloride concentration must
be high enough (preferably 325 mM for 2.5 mg/mL aPC and 650 mM for
5 mg/mL aPC formulations) to cause the sodium chloride to
crystallize-out during the freezing part of the freeze-drying
process. Formulations of aPC can be processed in a conventional
freeze dryer to produce lyophilized products consisting of 1 part
aPC, 6 parts sucrose, and 7.6 parts sodium chloride by weight.
EXAMPLE 2
Stability of aPC in Product Formulations Containing Different
Bulking Agents
[0049] Formulations of aPC were prepared to investigate the effect
of various bulking agents on the stability of the molecule. A total
of six excipients were added to aPC in phosphate buffer containing
no salt. These bulking agents are glycine, mannitol, sucrose,
trehalose, raffinose, and hydroxyethyl starch (HES). The stability
of aPC in the phosphate, no salt, no bulking agent formulation
("control") was compared to that in the bulking agent formulations.
Samples were stored at 50.degree. C., 40.degree. C., and 25.degree.
C. for various lengths of time. Data from analyses of these samples
were compared to the initial values (time=0). APTT potency, size
exclusion-high performance liquid chromatography (SE-HPLC),
SDS-PAGE, and protein content assays were used to evaluate the
physical and chemical stability of the formulations.
[0050] Formulations of aPC were prepared by dissolving aPC in
phosphate buffer to 5 mg/mL aPC. Bulking agents were added to
portions of the aPC solution at a ratio of 6:1 (bulking agents to
aPC), or 30 mg/mL. The samples were lyophilized to 5 mg
aPC/vial.
[0051] The formulations were put on stability at 50.degree. C. for
14 and 28 days; 40.degree. C. for 28 days, 48 days and 6 months;
and 25.degree. C. for 6 and 12 months. For each time point, two
vials of each formulation were analyzed independently as separate
samples and data from these samples were compared to those from
initial values (time=0). Analyses included aPC potency (APTT),
SDS-PAGE, percent of aPC monomer, and protein content.
2 25.degree. C. 50.degree. C. 40.degree. C. 6 12 14 28 28 84 6 Vial
Initial month month Initial day day Initial day day month control
APTT 1 321 294 236 321 248 248 321 248 221 215 Potency (U/mg) 2 321
251 242 321 245 227 321 279 233 176 Monomer 1 99.3 98.3 96.5 99.3
97.5 97.0 99.3 97.7 96.2 95.1 Content 2 99.2 95.8 96.4 99.2 97.3
97.1 99.2 97.7 96.1 95.4 (%) glycine APTT 1 282 233 142 282 164 97
282 191 155 158 Potency (U/mg) 2 321 239 191 321 161 142 321 215
152 79 Monomer 1 99.1 98.4 93.3 99.1 97.4 97.2 99.1 97.8 96.4 95.8
Content 2 99.1 98.4 96.3 99.1 97.3 97.1 99.1 97.7 96.4 95.7 (%)
mannitol APTT 1 309 227 255 309 270 245 309 273 270 282 Potency
(U/mg) 2 321 321 267 321 239 242 321 300 251 191 Monomer 1 99.2
98.8 97.4 99.2 98.2 98.1 99.2 98.4 97.6 97.8 Content 2 99.2 98.7
97.6 99.2 98.2 98.0 99.2 98.4 97.6 97.8 (%) sucrose APTT 1 327 300
288 327 300 288 327 267 306 285 Potency (U/mg) 2 297 300 306 297
291 291 297 321 242 294 Monomer 1 99.2 99.0 98.5 99.2 98.7 98.9
99.2 98.8 98.5 98.9 Content 2 99.2 99.0 98.5 99.2 98.7 98.9 99.2
98.8 98.5 98.9 (%) trehalose APTT 1 312 291 282 312 258 282 312 273
276 276 Potency (U/mg) 2 309 315 282 309 270 215 309 303 245 255
Monomer 1 99.2 99.0 98.4 99.2 98.6 98.8 99.2 98.8 98.4 98.7 Content
2 99.2 98.8 98.4 99.2 98.6 98.8 99.2 98.7 98.4 98.7 (%) raffinose
APTT 1 321 270 255 321 261 258 321 276 273 279 Potency (U/mg) 2 288
285 306 288 255 264 288 270 239 255 Monomer 1 99.1 99.0 97.0 99.1
98.6 98.7 99.1 98.7 98.4 98.6 Content 2 99.1 99.0 98.2 99.1 98.6
98.7 99.1 98.7 98.4 98.6 (%) HES APTT 1 282 188 176 282 182 164 282
194 185 145 Potency (U/mg) 2 285 245 215 285 188 161 285 176 152
103 Monomer 1 97.8 95.6 92.2 97.8 93.0 91.8 97.8 93.7 90.6 88.7
Content 2 97.8 95.3 91.8 97.8 92.9 91.0 97.8 92.9 90.5 88.5 (%)
[0052] There were no significant changes in pH, color, package
characteristics and physical appearance for any of the samples over
the one year stability time period. When analyzed by the APTT and
SE-HPLC procedures, the HES and glycine formulation had less
physical stability (through aggregation) and chemical stability
(potency) when compared to the control. The mannitol formulation
offered slightly better physical and chemical stability than the
control, and the remaining formulations, sucrose, trehalose and
raffinose, all demonstrated even more superior physical and
chemical stability when compared to the control. Therefore,
mannitol sucrose, trehalose and raffinose, as bulking agents in aPC
formulations, offer increased chemical and physical stability when
compared to an aPC formulation without a bulking agent or those
having glycine or HES.
EXAMPLE 3
Stability of Recombinant Human Activated Protein C
[0053] Two lots of a lyophilized formulation of recombinant human
activated protein C (aPC) were stored for 1 month at 40.degree.
C./75% relative humidity, and then analyzed for possible
degradation. The stability of aPC was also monitored after
reconstitution with sterile water and storage for up to 72 hours at
ambient temperature. The lyophilized aPC product consisted of 10 mg
aPC, 60 mg sucrose, 76 mg sodium chloride, and 15.1 mg citrate per
vial. The aPC in this formulation is stable in the dry state for at
least one month when stored at 40.degree. C./75% relative humidity,
and in solution for 24 hours when stored at ambient
temperature.
[0054] Both lots were prepared using the same unit formula of 10 mg
aPC, 60 mg sucrose, 76 mg sodium chloride, and 15.1 mg citrate per
vial. Both lyophilized lots of aPC were stored for 1 month at
40.degree. C./75% relative humidity and the stability of aPC was
monitored using the APTT potency assay, ion-pairing HPLC for
quantitation of aPC peptides and mass spectrometry for quantitation
of protein variant forms. One lot was also reconstituted with
sterile water, to 1 mg/mL aPC, and held at ambient temperature. The
stability of aPC in solution was monitored at the 0, 1, 4, 8, 24,
48 and 72 hour time points using the APTT and mass spectrometry
methods.
[0055] There was no loss of aPC activity and an insignificant
amount of structural degradation of the molecule after storage in
the dry state for one month at 40.degree. C./75% relative humidity.
The aPC in this formulation is stable for up to 24 hours at about 1
mg/mL to about 4 mg/mL after reconstitution.
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