PROTEIN FORMULATIONS WITH REDUCED VISCOSITY AND USES
THEREOF
TECHNICAL FIELD
The field relates to protein formulations and, more particularly, to protein
formulations with reduced, viscosity.
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to provisional U.S. Application Serial
No.60/752660, filed on December 21, 2005, which is herein incorporated by reference in
its entirety.
BACKGROUND
It is estimated that more than 371 new biotechnology-based medicines are in the
industry pipeline. Such biotechnology-based medicines include therapeutic proteins such
as enzymes, soluble receptors, ligands, blood proteins, and monoclonal antibodies.
Protein-based therapy, especially monoclonal antibody-based therapy, has become an
important method for treating diseases such as cancer, allergic diseases, asthma, and
organ transplantation. At the end of 2003 fourteen antibody-based therapies had been
approved by the Food and Drug Administration to treat different human diseases.
Antibody-based therapy is usually administered on a regular basis and requires
several mg/kg dosing by injection. Subcutaneous injection is a typical route of
administration of these therapies. Because of the small volumes used for subcutaneous
injection (usually 1.0 m.L-1.2 mL), for high dose antibody therapies, this route of
administration requires high concentration protein formulations (e.g., 50 mg/ml-300
mg/ml).
High protein concentrations pose challenges relating to the physical and chemical
stability of the protein, and difficulty with manufacture, storage, and delivery of the
protein formulation. One problem is the tendency of proteins to form particulates during
processing and/or storage, which make manipulation during further processing difficult.
To attempt to obviate this problem, surfactants and/or sugars have been added to protein
formulations. Although surfactants and sugars may reduce the degree of particulate

formation of proteins, they do not address another problem associated with manipulating
and administering concentrated protein formulations, i.e., increased viscosity. In fact,
sugars may enhance the intermolecular interactions within a protein or between proteins
and increase the viscosity of the protein formulation.
Increased viscosity of protein formulations has negative ramifications from
processing through drug delivery to the patient. Accordingly, there is a need in the art to
develop relatively high concentration protein formulations with suitably low viscosities
that are suitable for manufacture, storage, and administration.
SUMMARY
The instant application relates to protein formulations having reduced viscosity
compared to a corresponding protein formulation that does not include a viscosity-
reducing agent in a suitable concentration, and methods of making such protein
formulations having reduced viscosity (reduced viscosity formulations).
In one aspect, the invention relates to methods of reducing the viscosity of a
protein formulation by adding a viscosity reducing agent to a protein formulation, thereby
. reducing the viscosity of the protein formulation compared to a protein formulation
lacking the viscosity reducing agent. In one embodiment, the method involves
determining the viscosity of a protein formulation prior to the addition of a viscosity
reducing agent. In another embodiment, the method involves determining the viscosity of
a protein formulation after the addition of a viscosity reducing agent. In yet another
embodiment, the method involves determining the viscosity of a protein formulation prior
to and after the addition of a viscosity reducing agent In certain embodiments, the
viscosity reducing agent reduces the viscosity of the protein formulation by at least 5%
compared to the viscosity of the formulation formulated without the viscosity reducing
agent.
In some embodiments, the viscosity reducing agent is calcium chloride or
magnesium chloride. The viscosity reducing agent is added at low concentrations so as
not to negatively impact the protein formulation. The viscosity reducing agent is
generally added to a protein formulation to a final concentration of between about 1 mM
and about 50 mM. In some embodiments, the viscosity reducing agent is added to a
protein formulation to a final concentration of between about 5 mM and about 25 mM. In

certain embodiments, the viscosity reducing agent is added to a protein formulation to a
final concentration of between about 1 mM and about 20 mM. In certain embodiments,
the viscosity reducing agent is added to a protein formulation to a final concentration of
between 0.5 mM and 14 mM. In another embodiment, the protein is an antibody, an Ig
fusion protein, a receptor, a ligand, a transcription factor, an enzyme, or a biologically
active fragment thereof. In some embodiments, the protein is an anti-myostatin antibody,
an anti-IL-12 antibody, or an anti-IL-13 antibody.
In another aspect, the invention relates to a reduced viscosity protein formulation.
The reduced viscosity protein formulation includes a protein, a viscosity reducing agent,
and a buffer. In some embodiments, the viscosity reducing agent is calcium chloride or
magnesium chloride. The viscosity reducing agent is generally added to a protein
formulation to a final concentration of between about 1 mM and about 50 mM. In some
embodiments, the viscosity reducing agent is added to a protein formulation to a final
concentration of between about 5 mM and about 25 mM. In certain embodiments, the
viscosity reducing agent is added to a protein formulation, to a final concentration of
between about 1 mM and about 15 mM. In certain other embodiments, the viscosity
reducing agent is added to a protein formulation to a final concentration of between
0.5 mM and 14 mM. When the viscosity reducing agent is added to a protein formulation
to a concentration of between about 0.5 mM to about 50 mM, sodium chloride and
sodium biphosphate are not used as viscosity reducing agents. The pH of the protein
formulation is generally between about 5.5 and about 6.5. In certain embodiments, the
protein is an antibody, an Ig fusion protein, a receptor, a ligand, a transcription factor, an
enzyme, or a biologically active fragment thereof. In certain embodiments, the protein
formulations are provided as kits. Such kits can include instructions for use of the protein
formulation.
In certain embodiments, the reduced viscosity protein formulation is a reduced
viscosity anti-myostatin antibody formulation. In one embodiment, the anti-myostatin
antibody is a monoclonal antibody. In another embodiment, the anti-myostatin antibody
is a humanized monoclonal antibody (e.g., a partially humanized or fully humanized
monoclonal antibody). In certain embodiments, the anti-myostatin antibody is MYO-022,
MYO-028 or MYO-029. Anti-myostatin antibodies are generally used at a concentration
of between about 25 mg/ml to about 400 mg/ml. The viscosity reducing agent is

generally added to a reduced viscosity anti-myostatin antibody formulation to a final
concentration of between about 1 mM and about 50 mM. In some embodiments, the
viscosity reducing agent is added to an anti- myostatin antibody to a final concentration of
between about 5 mM and about 25 mM. In certain embodiments, the viscosity reducing
agent is added to an anti-myostatin antibody formulation to a final concentration of
between about 1 mM and about 15 mM. In certain embodiments, the viscosity reducing
agent is added to an anti-myostatin antibody formulation to a final concentration of
between 0.5 mM and 14 mM. When the viscosity reducing agent is added to an anti-
myostatin antibody formulation to a concentration of between about 0.5 mM to about
50 mM, sodium chloride and sodium biphosphate are not used as viscosity reducing
agents. Reduced viscosity anti-myostatin antibody formulations generally have a pH of
between about 5.5 and about 6.5. In one embodiment, histidine is used to buffer a
reduced viscosity myostatin antibody formulation. A reduced viscosity myostatin
antibody formulation can also include one or more cryoprotectants, one or more
surfactants, one or more anti-oxidants, or a combination thereof. In some embodiments,
the reduced viscosity anti-myostatin formulation is a reconstituted formulation.
Myostatin antibodies can be formulated as described herein as pharmaceutical
compositions and used to treat disorders such as, but not limited to, muscular dystrophy,
sarcopenia, cachexia, and Type II diabetes. In certain embodiments, a reduced viscosity
anti-myostatin antibody formulation is provided as a kit. Such kits can include
instructions for use of the antibody formulation.
In certain embodiments, the reduced viscosity protein formulation is a reduced
viscosity anti-IL-12 antibody formulation. In one embodiment, the anti-IL-12 antibody is
a monoclonal antibody. In another embodiment, the anti-IL-12 antibody is a humanized
monoclonal antibody (e.g., a partially humanized or fully humanized monoclonal
antibody). In certain embodiments, the anti-IL-12 antibody is J695. Anti-EL-12
antibodies are generally used in a formulation at a concentration of between about 25
mg/ml to about 400 mg/ml. A viscosity reducing agent is generally added to an anti-IL-
12 antibody formulation to a final concentration of between about 1 mM and about 50
mM. In some embodiments, the viscosity reducing agent is added to an anti-IL-12
antibody formulation to a final concentration of between about 5 mM and about 25 mM.
In certain embodiments, the viscosity reducing agent is added to an anti-IL-12 antibody

formulation to a final concentration of between about 1 mM and about 15 mM. In certain
other embodiments, the viscosity reducing agent is added to an anti-IL-12 antibody
formulation to a final concentration of between 0.5 mM and about 14 mM. When the
viscosity reducing agent is added to an. anti-IL-12 antibody formulation to a concentration
of between about 0.5 mM to about 50 mM, sodium chloride and sodium biphosphate are
not used as viscosity reducing agents. Reduced viscosity anti-IL-12 antibody
formulations generally have pH of between about 5.5 and about 6.5. In certain
embodiments, histidine is used as a buffer in a reduced viscosity IL-12 antibody
formulation. Reduced viscosity anti-IL-12 antibody formulations can also include one or
more cryoprotectants, one or more surfactants, one or more anti-oxidants, or
combinations thereof. In some embodiments, the reduced viscosity anti-IL-12
formulation is a reconstituted formulation. Anti-IL-12 antibodies can be formulated as
described herein for use as pharmaceutical compositions and used to treat disorders such
as, but not limited to, rheumatoid arthritis, Crohn's disease, psoriasis, and psoriatic
arthritis. In certain embodiments, a reduced viscosity anti-IL-12 antibody formulation is
provided as part of a kit. Such kits can include instructions for use of the anti-IL-12
antibody formulation.
In certain embodiments, the reduced viscosity protein formulation is an anti-IL-13
antibody formulation, hi one embodiment, the anti-IL-13 antibody is a monoclonal
antibody. In another embodiment, the anti-IL-13 antibody is a humanized monoclonal
antibody (e.g., partially humanized or fully humanized). In certain embodiments, the
anti-IL-13 antibody is IMA-638. Anti-IL-13 antibodies are generally used in a
formulation at a concentration of between about 25 nag/ml to about 400 mg/ml. A
viscosity reducing agent is generally added to make a reduced viscosity anti-IL-13
antibody formulation to a final concentration of between about 1 mM and about 50 mM.
In some embodiments, the viscosity reducing agent is added to an anti-IL-13 antibody
formulation to a final concentration of between about 5 mM and about 25 mM. In certain
embodiments, the viscosity reducing agent is added to an anti-IL-13 antibody formulation
to a final concentration of between about 1 mM and about 15 mM. In certain other
embodiments, the viscosity reducing agent is added to an anti-IL-13 antibody formulation
to a final concentration of between 0.5 mM and about 14 mM. When the viscosity
reducing agent is added to an anti-IL-13 antibody formulation to a concentration of

between about 0.5 mM to about 50 mM, sodium chloride and sodium biphosphate axe not
used as viscosity reducing agents. Reduced viscosity anti-IL-13 antibody formulations
generally have a pH of between about 5.5 and about 6.5. In one embodiment, histidine is
used as a buffer in a reduced viscosity IL-13 antibody formulation. A reduced viscosity
anti-IL-13 antibody formulation can also include one or more cryoprotectants, one or
more surfactants, one or more anti-oxidants, or combinations thereof. In some
embodiments, the reduced viscosity anti-IL-13 formulation is a reconstituted formulation.
Anti-IL-13 antibodies can be formulated in a reduced viscosity formulation as
pharmaceutical composition and used to treat disorders such as, but not limited to,
respiratory disorders (e.g., asthma); atopic disorders (e.g., allergic rhinitis); inflammatory
and/or autoimmune conditions of the skin (e.g., atopic dermatitis), gastrointestinal organs
(e.g., inflammatory bowel diseases (IBD)), as well as fibrotic and cancerous disorders. In
certain embodiments, a reduced viscosity anti-IL-13 antibody formulation is provided as a
kit Such kits can include instructions for use of the reduced viscosity anti-IL-13
antibody formulation.
Unless otherwise defined, all technical and scientific terms used herein have the
same meaning as commonly understood by one of ordinary skill in the art to which this
invention belongs. Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the present invention, suitable
methods and materials are described below. All publications, patent applications, patents,
and other references mentioned herein are incorporated by reference in their entirety. In
addition, the materials, methods, and examples are illustrative only and not intended to be
limiting.
Other features and advantages of the invention will be apparent from the detailed
description, drawings, and from the claims.
BRIEF DESCRIPTION OF THIS DRAWINGS
Fig. 1 is a graph depicting the results of experiments conducted to determine the
effect of increasing concentrations of various salts on the viscosity of an anti-myostatin
(MYO-029) antibody formulation.

Fig. 2 is a bar graph depicting the results of experiments conducted to determine
the effect of increasing concentrations of calcium chloride on the viscosity of an anti-
myostatin (MYO-028) antibody formulation.
Fig. 3 is a graph depicting the results of experiments conducted to determine the
effect of increasing concentrations of calcium chloride on the viscosity of an anti-IL-13
(IMA-638) antibody formulation.
Fig. 4A is a bar graph depicting the results of experiments conducted to test the
effect of freeze-thaw-induced degradation of the anti-myostatin (MYO-029) antibody in
the presence or absence of calcium chloride. Degradation was assessed by protein
recovery as determined by measuring absorbance at 280 run.
Fig. 4B is a bar graph depicting the results of experiments conducted to test the
effect of freeze-thaw-induced degradation of the anti-myostatin (MYO-029) antibody in
the presence or absence of calcium chloride. Degradation was assessed by percentage of
high molecular weight species (% HMW) formation as determined by size exclusion-high
performance liquid chromatography (SEC-HPLC).
Fig. 5 A is a bar graph depicting the results of experiments conducted to test the
effect of the presence or absence (control) of calcium chloride on the liquid stability of
anti-myostatin (MYO-029) antibody subjected to storage at 40°C for up to seven days.
Liquid stability of MYO-029 antibody was determined by measuring absorbance at 280
nm.
Fig. 5B is a bar graph depicting the results of experiments conducted to test the
effect of the presence or absence (control) of calcium chloride on the liquid stability of
anti-myostatin (MYO-029) antibody subjected to storage at 40°C for up to seven days.
Liquid stability of MYO-029 antibody was determined by measuring HMW formation as
determined by SEC-HPLC.
Fig. 6 is a representation of the amino acid sequence of the MYO-028 antibody
heavy chain (SEQ ID NO:1) and light chain (SEQ ID NO:2).
Fig. 7 is a representation of the amino acid sequence of the MYO-029 antibody
heavy chain (SEQ ID NO:3) and light chain (SEQ ID NO:4).
Fig. 8 is a representation of the amino acid sequence of the J695 antibody heavy
chain (SEQ ID NO:5) and light chain (SEQ ID NO:6).

Fig. 9 is a representation of the amino acid sequence of the IMA-638 antibody
heavy chain (SEQ ID NO:7) and light chain (SEQ ID NO:8). The last amino acid residue
encoded by the heavy chain DNA sequence, Lys448, is observed in the mature, secreted
form of IMA-638 only in small quantities and is presumably removed from the bulk of
the monoclonal antibody during intracellular processing by CHO cellular proteases.
Therefore, the carboxy-terminus of the IMA-638 heavy chain is Gly447. Carboxy-
terminus lysine processing has been observed in recombinant and plasma-derived
antibodies and does not appear to impact their function.
DETAILED DESCRIPTION
The viscosity of a protein formulation has implications for the stability,
processing, storage, and, for those used as drugs, drug delivery of the protein formulation
to a patient. Such implications include, but are not limited to concentration and buffer
exchange via ultrafiltration and diaifiltration (the flux across the membrane may decrease
with increasing viscosity thereby resulting in longer jprocessing times), sterile filtration, (it
takes longer to sterile filter viscous solutions, and in some instances a very viscous
solution will not pass through membranes with very small pores, e.g., 0.22 jim
membranes), sample handling (e.g., difficulty with pipetting and the ability to draw into a
syringe), recovery from the storage vial post reconstitution, stability, and passage through
needles for subcutaneous or intramuscular administration.
Provided herein are methods of reducing the viscosity of a protein formulation
that have been identified. The methods are suitable for preparing protein formulations
having reduced viscosity ("reduced viscosity formulations" or "reduced viscosity protein
formulations"). These reduced viscosity protein formulations include a protein of interest
and a viscosity reducing agent.
Methods of Reducing the Viscosity of a Protein Formulation
The term "viscosity" as used herein, may be "kinematic viscosity" or "absolute
viscosity." "Kinematic viscosity" is a measure of the resistive flow of a fluid under the
influence of gravity. When two fluids of equal volume are placed in identical capillary
viscometers and allowed to flow by gravity, a viscous fluid takes longer than a less
viscous fluid to flow through the capillary. If one fluid takes 100 seconds to complete its

flow and another fluid takes 200 seconds, the second fluid is twice as viscous as the first
on a kinematic viscosity scale. "Absolute viscosity," sometimes called "dynamic" or
"simple viscosity," is the product of kinematic viscosity and fluid density. The dimension
of kinematic viscosity is L2/T where L is a length and T is a time. Commonly, kinematic
viscosity is expressed in centistokes (cSt). The SI unit of kinematic viscosity is mm2/s,
which is 1 cSt. Absolute viscosity is expressed in units of centipoise (cP). The SI unit of
absolute viscosity is the milliPascal-second (mPa-s), where 1 cP=l mPa-s.
The viscosity of a protein formulation can be reduced by the addition of a
viscosity reducing agent to the formulation. In some cases, the viscosity reducing agent is
added at a relatively low concentration. The viscosity of a formulation comprising a
viscosity reducing agent is reduced compared to the viscosity of a formulation lacking the
viscosity reducing agent. When the addition of the viscosity reducing agent results in
lowering the viscosity of the formulation compared to a corresponding formulation that
does not include the viscosity reducing agent or compared to a formulation that does not
include the viscosity reducing agent at a selected concentration, the formulation
containing the viscosity reducing agent {e.g., in a selected concentration), the formulation
is a reduced viscosity formulation. In certain reduced viscosity formulations, the
viscosity reducing agent generally reduces the viscosity of a protein formulation by about
5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,
about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about
80%, about 85%, and about 90% compared to the viscosity of a protein formulation
without, or containing lower amounts of, the viscosity reducing agent. In some cases, the
viscosity reducing agent reduces the viscosity of a protein formulation by at least 5%, at
least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least
40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, and at least 90% compared to the viscosity of a
protein formulation without, or containing lower amounts of, the viscosity reducing agent.
In certain embodiments, the viscosity of a protein formulation is measured prior to the
addition of the viscosity reducing agent. In other embodiments, the viscosity of a protein
formulation is measured after the addition of the viscosity reducing agent. Such
measurements maybe made hours (e.g;, 1-23 hours), days (e.g., 1-10 days), weeks (e.g.,
1-5 weeks), or months (e.g., 1-12 months), or years (e.g., 1-2 years, 1-3 years) after the

addition of a viscosity reducing agent to a protein formulation. In yet other embodiments,
the viscosity of the protein formulation is measured prior to and after the addition of the
viscosity reducing agent Methods of measuring viscosity are well known in the art and
include, for example, using a capillary viscometer, or a cone-plate rheometer.
In one embodiment, the viscosity reducing agent is a salt such as calcium chloride,
magnesium chloride, sodium phosphate, or arginine hydrochloride. In the method
described herein, the viscosity reducing agent is added to the protein formulation to a
final concentration of between about 0.5 mM and about 100 rnM. In one embodiment,
the viscosity reducing agent is added to the protein formulation to a final concentration of
between about 5 mM and about 20 mM. In another embodiment, the viscosity reducing
agent is added to Ihe protein formulation to a final concentration of between 0.5 mM and
14 mM. Li certain embodiments, the viscosity reducing agent is added to the protein
formulation to a final concentration of between about 0.5 mM and not greater than 20
mM, or 19 mM, or 18 mM, or 17.imM, or 16 mM, or 15 mM, or 14 mM, or 13 mM, or 12
mM, or 11 mM, or 10 mM. In general, when the viscosity reducing agent is added to the
protein formulation to a final concentration of between about 0.5 mM and about 25 mM,
the viscosity reducing agent is calcium chloride or magnesium chloride, but not sodium
chloride, or sodium biphosphate. In certain embodiments, the viscosity reducing agent is
added at low concentrations so as not to negatively impact the protein formulation. For
example, at calcium chloride or magnesium chloride concentrations of 20 mM or greater,
proteins may form a gel at low storage temperatures (e.g,, 2-8°C). Accordingly, a
concentration of a viscosity reducing agent is generally selected for which the viscosity is
reduced at the intended storage temperature of the reduced viscosity formulation.
Formulations
The composition of a reduced viscosity protein formulation is determined by
consideration of several factors. These factors include, but are not limited to: the nature
of the protein (e.g., receptor, antibody, Ig fusion proteins, enzyme); the concentration of
the protein; the desired pH range; how the protein formulation is to be stored (e.g.,
temperature); the period of time over which the protein formulation is to be stored; and
how the formulation is to be administered to a patient. The selection of an appropriate

viscosity reducing agent is made based, in part, on such requirements for the protein in
the formulation.
Proteins
The protein of interest to be formulated includes, but is not limited to, proteins
such as, myostatin/GDF-8; interleukins (ILs), e.g., IL-1 to IL-15; growth hormones such
as human growth hormone and bovine growth hormone; growth hormone releasing
factor; parathyroid hormone; thyroid stimulating hormone; unease; bikunin; bilirubin
oxidase; subtilisin; lipoproteins; a-1-antitrypsin; insulin A-chain; insulin B-chain;
proinsulin;; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon;
Factor Vila; Factor VIII, Factor VIIIC; Factor IX; tissue factor; von Willebrand factor;
anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a
plasminogen activator, such as urokinase or tissue-type plasminogen activator (t-PA);
bombazine; thrombin; plasmin; miniplasmin; microplasmin; tumor necrosis factor-a and -
P; enkephalinase; RANTES (Regulated on Activation Normally T-cell Expressed and
Secreted); human macrophage inflammatory protein (MIP-1- a); serum albumin such as
human serum albumin; Mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain;
prorelaxin; mouse gonadotropin-associated peptide; DNase; inhibin; activin; vascular
endothelial growth factor (VEGF):; placental growth factor (P1GF); receptors for
hormones or growth factors; an integrin; protein A or protein D; rheumatoid factors; a
neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4,
-5, or -6 (NTS, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-p; platelet-
derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF;
epidermal growth factor (EGF); taansforming growtli factor (TGF) such as TGF-a and
TGF-, including TGF- 1, TGF- 2, TGF- 3, TGF- 4, or TGF- 5; insulin-like growth
factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I); insulin-like growth
factor binding proteins; CD proteins such as: CD2, CD3, CD4, CD8, CD9, CD19, CD20,
CD22, CD28, CD34, and CD45; erythropoietin (EPO); thrombopoietin (TPO);
osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an
interferon such as interferon-a, -, and -; a colony stimulating factor (CSF), e.g., M-
CSF, GM-CSF, and G-CSF; superoxide dismutase; T-cell receptors; members of the HER
receptor family such as the EGF receptor, HER2, HER3 or HER4 receptor; cell adhesion.

molecules such as LFA-1, VLA-4, ICAM-1, and VCAM; IgE; blood group antigens;
flk2/flt3 receptor; obesity (OB) receptor; decay accelerating factor (DAF); a viral antigen
such as, HIV gag, env, pol, tat, or rev proteins; homing receptors; addressins;
immunoadtiesins; and biologically active fragments, or variants of any of the above-listed
polypeptides. In some formulations, more than one type of protein or fragment is
included in the formulation.
The term "biologically active fragment" means a fragment of a protein that retains
at least one of the functions of the protein from which it is derived. A biologically active
fragment of an antibody includes an antigen-binding fragment of the antibody; a
biologically active fragment of a receptor includes a fragment of the receptor that can still
bind its ligand; a biologically active fragment of a ligand includes that portion of a ligand
that can still bind its receptor; and a biologically active fragment of an enzyme includes
that portion of the enzyme that can still catalyze a reaction catalyzed by the full length
enzyme. In one embodiment, a biologically active fragment retains at least about 5% of
the function of the protein from which it is derived. The function of a protein can be
assayed by methods known in the art (e.g., testing antibody-antigen interactions, testing
ligand-receptor interactions, testing enzymatic activity, testing transcriptional activity, or
testing DNA-protein interactions). In some cases, the fragment is a therapeutically useful
fragment, which may, for example, retain certain feattures of the protein from which it is
derived (e.g., binding to a specific ligand) but does not cause cellular response elicited by
the protein from which it is derived.
In certain embodiments, the protein to be formulated is an antibody. The antibody
may be one that can bind to one of the above-mentioned proteins. The term "antibody" as
used herein, includes polyclonal antibodies, monoclonal antibodies, antibody
compositions with polyepitope specificities, bispecific antibodies, diabodies, or other
purified preparations of antibodies and recombinant antibodies. The antibodies can be
whole antibodies, e.g., of any isotype (IgG, IgA, IgE, IgM, etc.), or fragments thereof,
which bind the antigen of interest. In a specific example of an antibody used in the
present invention, the antibody to be formulated is an antibody having the IgG isotype.
Antibodies can be fragmented using conventional or other techniques and the fragments
screened for binding to an antigen of interest. Generally, an antibody fragment comprises
the antigen-binding and/or the variable region of an intact antibody. Thus, the term

antibody fragment includes segments of proteolytically cleaved or recombinantly
prepared portions of an antibody molecule that are can selectively bind to a selected
protein. Non-limiting examples of such proteolytic and/or recombinant fragments include
Fab, F(ab')2, Fab1, Fv, and single chain antibodies (scFv) containing a V[L] and/or V[H]
domain joined by a peptide linker. The scFvs may be covalently or noncovalently linked
to form antibodies having two or more binding sites.
In some embodiments, the antibody is a humianized monoclonal antibody. The
term "humanized monoclonal antibody" as used herein, is a monoclonal antibody from a
non-human source (recipient) that has been altered to contain at least one or more of the
amino acid residues found in the equivalent human monoclonal antibody (donor). A
"fully humanized monoclonal antibody" is a monoclonal antibody from a non-human
source that has been altered to contain all of the amino acid residues found in the antigen-
binding region of the equivalent human monoclonal antibody. Humanized antibodies
may also comprise residues that are not found either in the recipient antibody or the donor
antibody. These modifications can be made to further refine and optimize antibody
functionality. A humanized antibody may also optionally comprise at least a portion of a
human immunoglobulin constant region (Fc). .
In certain embodiments, an antibody used in. a reduced viscosity formulation is an
anti-myostatin antibody (e.g., MYO-022, MYO-028 (Fig. 6), MYO-029 (Fig. 7)). MYO-
022, MYO-028, and MYO-029 antibodies are described in U.S. Patent Appl. No.
10/688,925 (Pub. No. 2004/0142382), which is incorporated herein by reference, hi other
embodiments, the antibody is an IL-12 antibody (e.g., J695 (Fig. 8)). The J695 antibody
is described in U.S. Pat. No. 6,914,128, which is incorporated herein by reference. In yet
another embodiment, the antibody is an anti-IL-13 antibody (e.g., IMA-638 (Fig. 9),
CAT-354). Anti-IL-13 antibodies are described in U.S. Patent Appl. No. 11/149,309,
which is incorporated herein by reference.
In some embodiments, the protein to be formulated is a fusion protein. In one
embodiment, the fusion protein is an immunoglobulin (Ig) fusion protein. In a specific
embodiment, the fusion protein comprises the IgG heavy chain constant region. In
another embodiment, the fusion protein comprises an amino acid sequence corresponding
to the hinge, CH2 and CH3 regions of human immunoglobulin C1. Examples of Ig

fusion proteins include CTLA4 Ig and VCAM2D-IgG. Methods of making fusion
proteins are known in the art (e.g., U.S. Patent Nos. 6,887,471 and 6,482,409).
In certain embodiments, the protein to be formulated is a protein that does not
include a Factor VII polypeptide, or anti-IgE antibody.
A reduced viscosity formulation can contain more than one protein as necessary
for the treatment of a particular disorder. The additional protein(s) typically have
complementary activities to the other protein(s) in the formulation, and do not adversely
affect the other protein(s) in the formulation. For example, it may be desirable to provide
a single formulation containing two or more antibodies that bind to myostatin; two or
more antibodies that bind to IL-12; or two or more antibodies that bind to IL-13. In
addition, a protein formulation can also contain non-protein substances that are of use in
the ultimate utility of the reduced viscosity protein formulation. For example, sucrose
can be added to enhance stability and solubility of the protein in solution; and histidine
can be added to provide appropriate buffer capacity. Such additional substances can be
part of a protein formulation prior to addition of a viscosity reducing agent or added in the
process for making a reduced viscosity formulation.
In certain embodiments, the protein to be formulated is essentially pure and/or
essentially homogeneous (i.e., substantially free from contaminating proteins, etc.) prior
to its use in the formulation. The term "essentially pure" protein means a composition
comprising at least about 90% by weight of a selected protein fraction, for example at
least about 95% by weight of the selected protein fraction. The term "essentially
homogeneous" protein means a composition comprising at least about 99% by weight of a
selected protein fraction, excluding the mass of various stabilizers and water in solution.
Concentration of the Protein in a Low Viscosity Formulation
The concentration of the protein in a reduced viscosity formulation is dependent
on the ultimate use of the formulation. Protein concentrations in the formulations
described herein are generally between about 10 mg/ml and about 300 mg/ml, e.g.,
between about 10 mg/ml and about 100 mg/ml, about 25 mg/ml and about 100 mg/ml,
about 50 mg/ml and about 100 mg/ml, about 75 mg/ml and about 100 mg/ml, about 100
mg/ml and about 200 mg/ml, about 125 mg/ml and about 200mg/ml, about 150 mg/ml
and about 200 mg/ml, about 200 mg/ml and about 300 mg/ml, and about 250 mg/ml and

about 300 mg/ml. For example, protein concentrations in the formulations described
herein can be between 10 mg/ml and 300 mg/ml, e.g., between 10 mg/ml and 100 mg/ml,
between 25 mg/ml and 100 mg/ml, between 50 mg/ml and 100 mg/ml, between 75 mg/ml
and 100 mg/ml, between 100 mg/ml and 200 mg/ml, between 125 mg/ml and 200mg/ml,
between 150 mg/ml and 200 mg/ml, between 200 mg/ml and 300 mg/ml, and between
250 mg/ml and 300 mg/ml. The term "between" is intended to be inclusive of the
minimal and maximal concentrations.
Reduced viscosity protein, formulations can be used for therapeutic purposes.
Accordingly, the concentration of the protein in a formulation used for a therapeutic
application is determined based on providing the protein in a dosage and volume that is
tolerated by, and of therapeutic value to, the patient. If a reduced viscosity formulation is
to be administered by injection, the protein concentration will be dependent on the
injection volume (usually 1.0 mL-1.2 mL). Protein based therapies can require several
mg/kg of dosing per week, per month, or per several months. Accordingly, if a protein is
to be provided at 2-3 mg/kg of body weight of the patient, and an average patient weighs
75 kg, 150 mg-225 mg of the protein will need to be delivered in a 1.0 mL-1.2 mL
injection volume. Alternatively, the formulation is provided in a concentration suitable
for delivery at more than one injection site per treatment.
As the concentration of the protein in a formulation increases, the viscosity of the
protein formulation is also likely to increase. Increased viscosity of the formulation
makes the formulation harder to administer. Accordingly, there is a need to decrease the
viscosity of protein formulations when the increased viscosity impacts its ability to be
utilized.
Viscosity Reducing Agents
It has been found that adding relatively low concentrations of certain viscosity
reducing agents to a protein formulation reduces the viscosity of the protein formulation.
The term "viscosity reducing agent" as used herein, includes any agent that reduces the
viscosity of a protein formulation compared to a protein formulation not containing, or
containing a lesser amount of, the viscosity reducing agent. For example, a viscosity
reducing agent generally reduces the viscosity of a protein formulation by about 5%,
about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about

90%, or about 95% compared to the viscosity of the protein formulation without, or
containing lower amounts of, a viscosity reducing agent. For example, a viscosity
reducing agent generally reduces the viscosity of the protein formulation by 5%, 10%,
20%, 30%, 40%, 50%, 60%, 70%, 90%, or 95% compared to the viscosity of a protein
formulation without, or containing lower amounts of, the viscosity reducing agent. Non-
limiting examples of viscosity reducing agents include calcium chloride, magnesium
chloride, sirginine hydrochloride, sodium chloride, sodium thiocyanate, ammonium
thiocyanate, ammonium sulphate, sodium phosphate, and ammonium chloride.
In one embodiment, the viscosity reducing agent is calcium chloride. In another
embodiment, the viscosity reducing agent is magnesium chloride. In an alternate
embodiment, more than one viscosity reducing agent is added to a protein formulation.
A viscosity reducing agent: is generally added to a protein formulation to a final
concentration of between 1 mM to about 150 mM, e..g., between about 1 mM and about
50 mM, between about 2 mM and about 40 mM, between about 3 mM and about 30 mM,
between about 4 mM and about 25 mM. between about 5 mM and about 20 mM, between
about 5 mM and about 25 mM, between about 5 mM and about 30 mM, between about 5
mM and eibout 40 mM, and between about 5 mM and about 50 mM. In certain
embodiments, the viscosity reducing agent is added to the protein formulation to a final
concentration of less than 14 mM., less than 13 mM, less than 12 mM, less than 11 mM,
less than 10 mM, less than 9 mM, less than 8 mM, less than 7 mM, less than 6 mM, less
than 5 mM, less than 4 mM, less than 3 mM, or less than 2 mM. In other embodiments,
the viscosity reducing agent is added to the protein formulation to a final concentration of
between 0.5 mM and 14 mM, between 0.5 mM and 13 mM, between 0.5 mM and 12 mM,
between 0.5 mM and 11 mM, between 0.5 mM and 10 mM, between 0.5 mM and 9 mM,
between 0.5 mM and 8 mM, between 0.5 mM and 7 mM, between 0.5 mM and 6 mM, or
between 0.5 mM and 5 mM. In one embodiment, trie viscosity reducing agent is calcium
chloride at a final concentration of between about 5 mM and about 20 mM in the
formulation. In another embodiment, the viscosity reducing agent is calcium chloride at a
final concentration of between 5 mM and about 14 mM in the formulation. In other
embodiments, the viscosity reducing agent is magnesium chloride at. a final concentration
of between about 5mM and about 20 mM in the formulation. In another embodiment, the

viscosity reducing agent is magnesium chloride at a final concentration of between 5 mM
and about 14 mM in the formulation.
The viscosity of a protein formulation can be measured by any suitable method
known in the art including, for example, using a capillary viscometer or a cone-plate
rheometer.
Buffers
The term "buffer" as used herein, includes those agents that maintain the pH of a
solution, e.g., a formulation, in a desired range. The pH of a formulation as described
herein is generally between about pH 5.0 to about 9.0, for example, about pH 5.5 to about
6.5, about pH 5.5 to about 6.0, about pH 6.0 to about 6.5, pH 5.5, pH 6.0, or pH 6.5. In
general, a buffer that can maintain a solution at pH 5.5 to 6.5 is used. Non-limiting
examples of buffers that can be used in a formulation described herein include, histidine,
succinate, ghxconate, tris (trometamol), phosphate, citrate, 2-morpholinoethanesulfonic
acid (MES), sodium phosphate, sodium acetate, and cacodylate.
Histidine is a buffer that is typically in reduced viscosity formulations that are to
be administered by subcutaneous, intramuscular, or peritoneal injection. The
concentration of the buffer is between about 5 mM and 30 mM. In one embodiment, the
buffer of a formulation is histidine at a concentration of about 5 mM to about 20 mM.
Excipients
In addition to the protein, a viscosity reducing agent, and buffer, a reduced
viscosity formulation as described herein may also contain other substances. Such
substances include, but are not limited to, cryoprotectants, lyoprotectants, surfactants,
bulking agents, anti-oxidants, and stabilizing agents. In one embodiment, a reduced
viscosity protein formulation described herein includes an excipient selected from the
group consisting of a cryoprotectant, a lyoprotectant, a surfactant, a bulking agent, an
anti-oxidant, a stabilizing agent, and combinations thereof.
The terra "cryoprotectant" as used herein, includes agents that provide stability to
the protein in a formulation against freezing-induced stresses, e.g.. by being preferentially
excluded from the protein surface. Cryoprotectants may also offer protection during
primary and secondary drying and long-term product storage. Non-limiting examples of

cryoprotectants include sugars, such as sucrose, glucose, trehalose, mannitol, mannose,
and lactose; polymers, such as dextran, hydroxyethyl starch and polyethylene glycol;
surfactants, such as polysorbates (e.g.. PS-20 or PS-80); and amino acids, such as glycine,
arginine, leucine, and serine. A cryoprotectant exhibiting low toxicity in biological
systems is generally used. The cryoprotectant, if included in the formulation, is generally
added to a final concentration of between about 0.1% and about 10% (weight/volume),
e.g., between about 0.5% and about 10%, between about 0.5% and about 5%, between
about 0.5% and about 2%, between about 1% and about 5%, or between about 5% and
about 10%.. In one embodiment, the cryoprotectant is sucrose at a concentration of
between about 0.5% and about 10% (weight/volume).
In one embodiment, a lyoprotectant is added to a formulation described herein.
The term "lyoprotectant" as used herein, includes agents that provide stability to the
protein during the freeze-drying or dehydration process (primary and secondary freeze-
drying cycles), e.g., by providing an amorphous glassy matrix and by binding with the
protein through hydrogen bonding, replacing the water molecules that are removed during
the drying process. This helps to maintain the protein conformation, minimize protein
degradation during the lyophilization cycle, and improve the long-term product stability.
Non-limiting examples of lyoprotectants include sugars, such as sucrose or trehalose; an
amino acid., such as monosodium glutamate, non-crystalline glycine or histidine; a
methylamine such, as betaine; a lyotropic salt, such as magnesium sulfate; a polyol, such
as trihydric or higher sugar alcohols, e.g., glycerin, erythritol, glycerol, arabitol, xylitol,
sorbitol, and mannitol; propylene glycol; polyethylene glycol; pluronics; and
combinations thereof. The amount of lyoprotectant added to a formulation is generally an
amount that does not lead to an unacceptable amount of degradation/aggregation of the
protein when the protein formulation is lyophilized. Where the lyoprotectant is a sugar
(such as sucrose or trehalose) and the protein is an antibody, non-limiting examples of
lyoprotectant concentrations in a reduced viscosity protein formulation are from about 10
mM to about 400 mM, from about 30 mM to about 300 mM, and from about 50 mM to
about 100 mM.
In certain embodiments, a surfactant is included in a formulation described herein.
The term "surfactant" as used herein, includes agents that reduce the surface tension of a
liquid by adsorption at the air-liquid interface. Examples of surfactants include, without

limitation, nonionic surfactants, such as polysorbates (e.g., polysorbate 80 or polysorbate
20); poloxamers (e.g., poloxamer 188); Triton™ (e.g.,Triton™X-100); sodium dodecyl
sulfate (SDS); sodium octyl glycoside; lauryl-sulfobetaine; myristyl-sulfobetaine;
linoleyl-sulfobetaine; stearyl-sulfobetaine; lauryi-sarcosine; myristyl-sarcosine; linoleyl-
sarcosine; stearyl-sarcosine; linoleyl-betaine; myristyl- betaine; cetyl-betaine;
lauroamidopropyl-betaine; cocamidopropyl-betaine; linoleamidopropyl-betaine;
myristamidopropyl-betaine, palmidopropyl-betaine; isostearamidopropyl-betaine (e.g.,
lauroamidopropyl); myristarnidopropyl-, pahnidopropyl-, or isostearamidopropyl-
dimethylamine; sodium methyl cocoyl-, or disodium methyl ofeyl-taurate; and the
Monaquat™ series (Mona Industries, Inc., Paterson, N.J.); polyethyl glycol; polypropyl
glycol; and copolymers of ethylene and propylene glycol (e.g., pluronics, PF68). The
amount of surfactant added is such that it maintains aggregation of the reconstituted
protein at an acceptable level as assayed using, e.g., SEC-HPLC to determine the
percentage of high molecular weight (HMW) species or low molecular weight (LMW)
species, and minimizes the formation of particulates after reconstitution of a lyophilate of
a protein formulation described herein. For example, fee surfactant can be present in a
formulation (liquid or prior to lyophilization) in an amount from about 0.001-0.5%, e.g.,
from about 0.05-0.3%.
In some embodiments, a bulking agent is included in a reduced viscosity
formulation. The term "bulking agent" as used herein, includes agents that provide the
structure of the freeze-dried product without interacting directly with the pharmaceutical
product. In addition to providing a pharmaceutically elegant cake, bulking agents may
also impart useful qualities in regard to modifying the collapse temperature, providing
freeze-thaw protection, and enhancing the protein stability over long-term storage. Non-
limiting examples of bulking agents include mannitol, glycine, lactose, and sucrose.
Bulking agents may be crystalline (such as glycine, mannitol, or sodium chloride) or
amorphous (such as dextran or hydroxyethyl starch) and are generally used in protein
formulations in an amount from 0.5% to 10%.
Other pharmaceutically acceptable carriers, excipients, or stabilizers, such as those
described in Remington: The Science and Practice of Pharmacy 20th edition, Gennaro,
Ed., Lippincott Williams & Wilkins (2000) may also be included in a protein formulation
described herein, provided that they do not adversely affect the desired characteristics of

the formulation. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients
(e.g., patients) at the dosages and concentrations employed and include: additional
buffering agents; preservatives; co-solvents; antioxidaints, including ascorbic acid and
methionine; chelating agents such as EDTA; metal complexes (e.g., Zn-protein
complexes); biodegradable polymers, such as polyesters; salt-forming counterfoils, such
as sodium, polyhydric sugar alcohols; amino acids, such as alanine, glycine, glutamine,
asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid,
and threonine; organic sugars or sugar alcohols, such as lactitol, stachyose, mannose,
sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol,
cyclitols (e.g., inositol), polyethylene glycol; sulfur containing reducing agents, such as
urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, ct-monothioglycerol,
and sodium; thio sulfate; low molecular weight proteins, such as human serum albumin,
bovine serum albumin, gelatin, or other immunoglobulins; and hydrophilic polymers,
such as polyvinylpyrrolidone.
Exemplary Protein Formulations
MYO-029
In one example, a MYO-029 reduced viscosity formulation can be formulated
using 1 mg/ml to 300 mg/ml of the MYO-029 antibody. The MYO-029 formulation
generally includes between about 1 mM and about 50 mM calcium chloride or
magnesium, chloride. The formulation can include about 5 mM to about 25 mM histidine.
The formulation can include about 1% to about 5% (w/v) sucrose or trehalose. In some
instances, the formulation can include about 10 mM to about 25 mM methionine. In
certain MYO-029 formulations, 0.05-0.2% (w/v) polysorbate-20 or polysorbate-80 is
added. The pH of the formulation is generally between 5.5 and 6.5. In a specific
example, the MYO-029 formulation comprises 150 mg/ml of the MYO-029 antibody, 10
mM calcium chloride or magnesium chloride, 20 mM histidine, 4% sucrose, and has a pH
of 6.0. In another specific example, the MYO-029 formulation comprises 75 mg/ml of
the MYO-029 antibody, 5 mM calcium chloride or magnesium chloride, 10 mM histidine,
10 mM methionine, 2% sucrose, and has a pH of 6.0. In another specific example, a
MYO-029 antibody formulation comprises 150 mg/ml of the MYO-029 antibody, 10 mM

calcium chloride or magnesium chloride, 20 mM histidine, 20 mM methionine,
4% sucrose, 0.2% polysorbate-80, and has a pH of 6.0.
MYO-028
MYO-028 reduced viscosity formulations can be formulated using 1 mg/ml to 300
mg/ml of the MYO-028 antibody. The MYO-028 formulation generally includes
between about 1 mM and about 50 rnM calcium chloride or magnesium chloride. The
formulation can include between about 5 mM to about 25 mM histidine. The formulation
can include between about 1% to about 5% (w/v) sucrose or trehalose. The pH of a
MYO-028 formulation is generally between about 5.5 and about 6.5. In one specific
example, a MYO-028 antibody formulation comprises 50 hs mg/ml of the antibody, 10
mM histidine, 5% sucrose, and has a pH of 6.5. In another specific example, a MYO-028
antibody formulation comprises 50 ing/ml of the antibody, 10 mM calcium chloride or
magnesium chloride, 10 mM histidine, 5% sucrose, and has a pH of 6.5.
J69S
J695 reduced viscosity formulations can be formulated using 1 mg/ml to
300 mg/ml of the J695 antibody. A J695 formulation generally includes between about 1
mM and about 50 mM calcium chloride or magnesium chloride. The formulation can
include about 5 mM to about 25 mM histidine. The formulation may include about 1% to
about 5% (w/v) sucrose or trehalose. In some instances, the formulation can include
about 10 mM to about 25 mM methionine. In certain J695 formulations, between about
1% to about 5% (w/v) mannitol is added. The pH of the formulation is generally between
5.5 and 6.5. In a specific example, a J695 antibody formulation comprises 100 mg/ml of
the J695 antibody, 10 mM histidine., 10 mM methionine, 4% mannitol, 1% sucrose, and
has a pH of 6.0. In another specific example, a J695 antibody formulation comprises 100
mg/ml of the J695 antibody, 10 mM: histidine, 10 mM methionine, 5mM calcium chloride
or magnesium chloride, 4% mannitol, 1% sucrose, and has a pH of 6.0. In another
specific embpdiment, the J695 antibody formulation comprises 100 mg/ml of the J695
antibody, 10 mM histidine, 10 mM methionine, 10 mM calcium chloride or magnesium
chloride, 4% mannitol, 1% sucrose, and has a pH of 6.0.

IMA-638
IMA-638 protein formulations can be formulated using 1 mg/ml to 300 mg/ml of
the IMA-638 antibody. A reduced viscosity formulation containing IMA-638 generally
includes between about 1 mM and about 50 mM calcium chloride or magnesium chloride.
The formulation can include about 5 mM to about 25 mM histidine. The formulation can
also include about 1% to about 10% (w/y) sucrose or trehalose. The pH of the
formulation is generally between 5.5 and 6.5. In a specific example, the IMA-638
antibody formulation comprises 50 mg/ml of the IMA-638 antibody, 10 mM histidine,
5% sucrose, and has a pH of 6.0. In another specific example, the IMA-638 antibody
formulation comprises 100 mg/ml of the IMA-638 antibody, 20 mM histidine, 10%
sucrose, and has a pH of 6.0. In another specific example, the IMA-638 antibody
formulation comprises 50 mg/ml of the IMA-638 antibody, 5 mM calcium chloride or
magnesium, chloride, 10 mM histidine, 10% sucrose, and has a pH of 6.0. In yet another
specific exzimple, the IMA-638 antibody formulation comprises 100 mg/ml of the IMA-
638 antibody, 10 mM calcium chloride or magnesium chloride, 20 mM histidine, 10%
sucrose, and has a pH of 6.0.
Storage Methods
A reduced viscosity protein formulation described herein may be stored by any
suitable method known to one of skill in the art. Non-limiting examples of methods for
preparing a reduced viscosity formulation for storage include freezing, lyophilizing, and
spray drying the protein formulation.
In some cases, a reduced viscosity formulation is frozen for storage. Accordingly,
it is desirable that the formulation be relatively stable under such conditions, including
when subjected to freeze-thaw cycles. One method of determining the suitability of a
formulation for frozen storage is to subject a sample formulation to at least two, e.g.,
three to ten cycles of freezing (at, for example -20°C or -80°C) and thawing (for example
. by fast thaw at room temperature or slow thaw on ice), determining the amount of LMW
species and/or HMW species that accumulate after the freeze-thaw cycles and comparing
it to the amount of LMW species or HMW species present in the sample prior to the
freeze-thaw procedure. An increase in the LMW species or HMW species indicates
decreased stability of a protein stored as part of the formulation. Size exclusion high

performance liquid chromatography (SEC-HPLC) can be "used to determine the presence
of LMW and HMW species. A suitable formulation may accumulate undesirable HMW
species or LMW species, but not to the extent that the presence of the HMW species or
LMW species make the formulation unsuitable for its intended use.
In some cases, a formulation is stored as a liquid. Accordingly, it is desirable that
the liquid formulation be relatively stable under such conditions, including at various
temperatures. One method of determining the suitability of a formulation for liquid
storage is to store the sample formulation at several temperatures (such as 2-8°C, 15°C,
20°C, 25°C, 30°C, 35°C, 40°C, and 50°C) and monitoring the amount (e.g., change in
percentage) of HMW species and/or LMW species that accumulate over time.
Additionally, the charge profile of the protein may be monitored by cation exchange-high
performance liquid chromatography (CEX-HPLC).
In general, the percentage of high molecular weight species or low molecular
weight species is determined either as a percentage of the total protein content in a
formulation or as a change in the percentage increase over time (i.e., during storage), as is
appropriates for the assay and parameter being determined. In general, and in non-limiting
examples, the change in the percentage of protein in high molecular weight species or low
molecular weight species in a reduced viscosity formulation is not greater than 1Q%, e.g.,
not greater than about 8%, not greater than about 5%, or not greater than about 3% with
respect to the assayed parameter (eg., time, temperature, additional compounds in the
formulation, lyophilization, or shaking).
Alternatively, a formulation can be stored after lyophilization. The term
"lyophilization" as used herein, refers to a process by which the material to be dried is
first frozen followed by removal of the ice or frozen solvent by sublimation in a vacuum
environment. An excipient (e.g., lyoprotectant) maybe included in formulations that are
to be lyophilized so as to enhance stability of the lyophilized product upon storage. The
term "reconstituted formulation" as used herein, refers to a formulation that has been
prepared by dissolving a lyophilized protein formulation in a diluent such that the protein
is dispersed in the diluent. The term "diluent" as used herein, is a substance that is
pharmaceutically acceptable (safe and non-toxic for administration to a human) and is
useful for the preparation of a liquid formulation, such as a formulation reconstituted after
lyophilization. Non-limiting examples of diluents include sterile water, bacteriostatic

water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline),
sterile saline solution, Ringer's solution, dextrose solution, or aqueous solutions of salts
and/or buffers.
Testing a reduced viscosity formulation for the stability of the protein component
of the formulation after lyophilization is useful for determining the suitability of a
formulation. The method is similar to that described above for freezing, except that the
sample formulation is lyophilized instead of frozen, reconstituted using a diluent and the
reconstituted formulation is tested for the presence of LMW species and/or HMW
species. An increase in LMW species or HMW species in the lyophilized sample
compared to a corresponding sample formulation that was not lyophilized indicates
decreased stability in the lyophilized sample.
In some cases, a formulation is spray-dried and then stored. For spray drying, a
liquid formulation is aerosolized in the presence of a dry gas stream. Water is removed
from the formulation droplets into the gas stream, resulting in dried particles of the drug
formulation. Excipients may be included in the formulation to (1) protect the protein
during the spray-drying dehydration, (2) protect the protein during storage after spray
drying, and/or (3) give the solution properties suitable for aerosolization. The method is
similar to that described above for freezing, except that the sample formulation is spray-
dried instead of frozen, reconstituted in a diluent and the reconstituted formulation is
tested for the presence of LMW species and/or HMW species. An increase in LMW or
HMW species in the spray-dried sample compared to a corresponding sample formulation
that was not lyophilized indicates decreased stability in the spray-dried sample.
Methods of Treatment
The reduced viscosity formulations described herein are useful as pharmaceutical
compositions in the treatment and/or prevention of a disease or disorder in a patient in
need thereof. The term "treatment" refers to both therapeutic treatment and prophylactic
or preventative treatment. Treatment includes the application or administration of the
reduced viscosity formulation to the body, an isolated tissue, or cell from a patient who
has a disorder, a symptom of a disorder, is at risk for a disorder, or a predisposition
toward a disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy,
ameliorate, improve, or affect the disorder, the symptom of the disorder, or the

predisposition toward the disorder. Those "in need of treatment" include those who
already have a disorder, as well as those in whom a disorder is to be prevented. The term
"disorder" is any condition that would benefit from treatment with a protein formulation
described herein. This includes chronic and acute disorders or diseases including those
pathological conditions that predispose the subject (partient) to the disorder in question.
Non-limiting examples of disorders to be treated using a formulation described herein
include autoimmune disorders, inflammatory disorders, muscle wasting disorders,
allergies, cancers, muscular dystrophy, sarcopenia, cachexia, Type II diabetes,
rheumatoid arthritis, Crohn's disease, psoriasis, psoriatic arthritis, asthma, dermatitis,
allergic rhinitis, chronic obstructive pulmonary disease, eosinophilia, fibrosis, and excess
mucus production.
In one embodiment, the reduced viscosity formulation suitable for use as a
pharmaceutical composition comprises an anti-myostatin antibody and a viscosity
reducing agent. In one embodiment, the anti-myostatin antibody is MYO-029. In other
embodiments, the anti-myostatin antibody is MYO-022 or MYO-028. The anti-myostatin
antibody is generally at a concentration of between about 0.5 mg/ml and about 300 mg/ml
in the formulation. In another embodiment, the viscosity reducing agent is at a final
concentration of between about 0.5 mM and 20 mM in the pharmaceutical composition.
In another embodiment, the viscosity reducing agent is at a final concentration of between
about 0.5 mM and 14 mM in the pharmaceutical composition. In another embodiment,
the pharmaceutical composition comprises an anti-myostatin antibody, a viscosity
reducing agent, and a buffer wherein the pH of the formulation is between about 5.5 to
about 6.5. The pharmaceutical compositions described herein may also contain other
proteins, drugs, and/or excipients. In particular, other proteins or substances useful for
treating the disorder in question may be added to the formulation. Anti-myostatin
antibody- containing pharmaceutical compositions are useful in the treatment or
prevention of disorders such as, but not limited to, muscle wasting disorders, muscular
dystrophy, sarcopenia, cachexia, and Type II diabetes.
In another embodiment, a pharmaceutical composition comprises an anti-IL-12
antibody and a viscosity reducing agent. In one embodiment, the anti-IL-12 antibody is
J695. The anti-IL-12 antibody is generally at a concentration of between about 0.5 mg/ml
and about 300 mg/ml in the formulation. In another embodiment, the viscosity reducing

agent is at a final concentration of between about 0.5 mM and 20 mM in the
pharmaceutical composition. In another embodiment, the viscosity reducing agent is at a
final concentration of between about 0.5 mM and 14 mM in the pharmaceutical
composition. In another embodiment, the pharmaceutical composition comprises an anti-
1L-12 antibody, a viscosity reducing agent, and a buffer, wherein the pH of the
formulation, is between about 5.5 to about 6.5. The pharmaceutical compositions
described herein may also contain other proteins, drugs, and/or excipients. In particular,
other proteins or substances useful for treating the disorder in question may be added to
the formulation. Anti-IL-12 antibody containing pharmaceutical compositions are useful
in the treatment or prevention of disorders such as, but not limited to, autoimmune
disorders, inflammatory disorders, rheumatoid arthritis, Crohn's disease, psoriasis, and
psoriatic arthritis.
In another embodiment, a pharmaceutical composition comprises an anti-IL-13
antibody and a viscosity reducing agent. In one embodiment, the anti-IL-13 antibody is
IM A-63 8. 'The anti-IL-13 antibody is generally at a concentration of between about 0.5
mg/ml and about 300 mg/ml in the formulation. In another embodiment, the viscosity
reducing agent is at a final concentration of between about 0.5 mM and 20 mM in the
pharmaceutical composition. In another embodiment, the viscosity reducing agent is at a
final concentration of between about 0.5 mM and 14 mM in the pharmaceutical
composition. In another embodiment, the pharmaceutical composition comprises an anti-
IL-13 antibody, a viscosity reducing agent, and a buffer wherein the pH of the
formulation is between about 5.5 to about 6.5. The pharmaceutical compositions
described herein may also contain other proteins, drugs, and/or excipients. In particular,
other proteins or substances useful for treating the disorder in question may be added to
the formulation. Anti-IL-13 antibody containing pharmaceutical compositions are useful
in the treatment or prevention of disorders such as, but not limited to, asthmatic disorders,
atopic disorders, chronic obstructive pulmonary disease, conditions involving airway
inflammation, eosinophilia, fibrosis and excess mucus production, inflammatory
conditions, autoimmune conditions, tumors or cancers, and viral infection.

Administration
A reduced viscosity formulation described herein can be administered to a subject
in need of treatment using methods known in the art, such as by single or multiple bolus
or infusion over a long period of time in a suitable manner, e.g., injection or infusion by
subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or
intraarticular routes, topical administration, inhalation, or by sustained release or
extended-release means. If the formulation has been lyophilized, the lyophilized material
is first reconstituted in an appropriate liquid prior to administration. The lyophilized
material can be reconstituted in, e.g., BWFI, phosphate buffered saline, or the same
formulation the protein had been in prior to lyophilization.
Parenteral compositions can be prepared in dosage unit form for ease of
administration and uniformity of dosage. "Dosage unit form" as used herein, refers to
physically discrete units suited as unitary dosages for the subject to be treated; each unit
containing a predetermined quantity of active compound calculated to produce the desired
therapeutic effect in association with the selected pharmaceutical carrier.
In the case of an inhalation method, such as metered dose inhaler, the device is
designed to deliver an appropriate amount of a formulation. For administration by
inhalation, the compounds are delivered in the form of an aerosol spray from a pressured
container or dispenser that contains a suitable propellant, e.g., a gas, such as carbon
dioxide, or a nebulizer. Alternatively, an inhaled dosage form may be provided as a dry
powder using a dry powder inhaler.
A reduced viscosity forrnuktion can also be entrapped in microcapsules prepared,
for example, by coacervation techniques or by interfacial polymerization, for example,
hydroxyrnethylcelhilose or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes,
albumin miicrospheres, microemulsiions, nanoparticles, and nanocapsules) or in
macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences,
20th edition {supra).
Sustained-release preparations of the protein formulations described herein can
also be prepared. Suitable examples of sustained-release preparations include
semipermeable matrices of solid hydrophobic polymers containing the protein
formulation. Examples of sustained-release matrices include polyesters, hydrogels (for

example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol))3 polylactides,
copolymers of L-glutamic acid and y-ethyi-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymeirs, and poly-D-(-)r3-hydroxybutyric
acid. The sustained-release formulations of the proteins described herein can be
developed using e.g., polylactic-coglycolic acid (PLGA) polymer due to its
biocompatibility and wide range of biodegradable properties. The degradation products
of PLGA, lactic and glycolic acids, can be cleared quickly within the human body.
Moreover, the degradability of this polymer can be adjusted from months to years
depending on its molecular weight and composition. Liposomal compositions can also be
used to formulate the proteins or antibodies disclosed herein.
Dosing
Toxicity and therapeutic efficacy of a formulation can be determined by
pharmaceutical procedures known in the art using, for example, cell cultures or
experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the
population) and the ED50 (the dose therapeutically effective in 50% of the population).
The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be
expressed as the ratio LD50/ED50.
The data obtained from the cell culture assays and animal studies can be used in
formulating a range of dosage for use in humans. The dosage of such formulations
generally lies within a range of circulating concentrations that include the ED50 with little
or no toxicity. The dosage may vary within this range depending upon the dosage form
employed and the route of administration utilized. For any formulation used in the
method of the invention, the therapeutically effective dose can be estimated initially from
cell culture assays. A dose can be formulated in animal models to achieve a circulating
plasma concentration range that includes the IC50 (i.e., the concentration of the test
compound which achieves a half-maximal inhibition of symptoms) as determined in cell
culture. Such information can be used to more accurately determine useful doses in
humans. Levels in plasma may be measured, for example, by high performance liquid
chromatography.
The appropriate dosage of the protein of the formulation will depend on the type
of disorder to be treated, the severity and course of the disorder, whether the agent is

administered for preventive or therapeutic purposes, previous therapy, the patient's
clinical history and response to the agent, and the discretion of the attending physician. A
formulation is generally delivered such that the dosage is between about 0.1 mg
protein/kg of body weight to 100 mg protein/kg of body weight. The formulation is
administered to the patient at one time or over a series of treatments. In one embodiment,
amyostatin antibody (e.g., MYO-22, MYO-28, MYO-029) formulation is delivered to a
patient in need thereof at a dosage of 1 mg/kg to 10 mg/kg of body weight. In another
embodiment, an IL-12 antibody formulation is administered to a patient in need thereof at
a dosage of 1 mg/kg to 5 mg/kg of body weight. In a further embodiment, an IL-13
antibody formulation is administered to a patient in need thereof at a dosage of about 0.5
mg/kg to about 5 mg/kg of body weight of the patient..
A formulation to be used for in vivo administration must be sterile. A formulation
can be rendered sterile for example, by filtration through sterile filtration membranes,
prior to, or following, formulation of a liquid or lyophilization and reconstitution. The
therapeutic compositions disclosed herein generally are placed into a container having a
sterile access port, for example, an intravenous solution bag, or vial having a stopper
pierceable by a hypodermic injection needle.
Articles of Manufacture
In another embodiment, an article of manufacture is provided that contains a
formulation described herein and typically provides instructions for its use. The article of
manufacture comprises a container suitable for containing the formulation. Suitable
containers include, without limitation, bottles, vials (e.g., dual chamber vials), syringes
(e.g., single or dual chamber syringes), test tubes, nebulizers, inhalers (e.g., metered dose
inhalers or dry powder inhalers), or depots. The container can be formed from a variety
of materials, such as glass, metal or plastic (e.g., polycarbonate, polystyrene,
polypropylene). The container holds the formulation and the label on, or associated with,
the container can indicate directions for reconstitution and/or use. The label may further
indicate that the formulation is useful or intended for subcutaneous administration. The
container holding the formulation may be a multi-use; vial, which allows for repeat
administrations (e.g., from 2-6 doses) of the formulation. The article of manufacture may
further comprise a second container comprising a suitable diluent (e.g., WFI, 0.9% NaCl,

BWFI, or phosphate buffered saline). When the article of manufacture comprises a
lyophilized version of a protein formulation, mixing of a diluent with the lyophilized
formulation will provide a final protein concentration in the reconstituted formulation of
generally at least 20 mg/ml. The article of manufacture may further include other
materials desirable from a commercial and user standpoint, including other buffers,
diluents, filters, needles, syringes, and package inserts with instructions for use.
The invention is further illustrated by the following examples. The examples are
provided for illustrative purposes only. They are not to be construed as limiting the scope
or content of the invention in any way.
EXAMPLES
Example 1
Viscosity of Antibody Formulations
Anti-p-amyloid peptide (anti-AB), anti-IL-13, anti-IL-12 (J695) and ahti-
myostatin (MYO-029) antibodies were formulated as described in Table 1. The viscosity
of these antibody formulations was measured using an Anton Paar Physica MCR301 cone
and plate rheometer. Specifically, a CP25-1 (24.971 mm diameter, 1.002° angle) cone
was used for all of the measurements; the shear rate was constant at 898 1/s for a duration
of 100 seconds. Measurements were made every 10 seconds. The viscosity
measurements were performed at both 4°C and 25°C using a built-in Peltier temperature
control unit. The liquid sample load on the plate was 90 ul. Each sample was analyzed in
triplicate.
Table 1 below lists the viscosities of different antibodies at different
concentrations and in different formulations.


The data shown in Table 1 demonstrate that the viscosity of anti-myostatin
(MYO-029) is significantly higher compared to the other antibodies listed in the Table.
The viscosities of all of the antibodies increased at 4°C. This increase is proportionally
much higher for MYO-029.
Example 2
Effect of Various Salts on the Viscosity of an MYO-029 Antibody Formulation
MYO-029 antibody, at a concentration of 73 nag/ml, was formulated in 10 mM
histidine, 2% sucrose, pH 6.0. Concentrated solutions of salts (e.g., calcium chloride,
magnesium chloride, sodium chloride, and sodium biphosphate) were diluted into the
MYO-029 antibody formulation using a pipette. The effect of these salts on the viscosity
of MYO-029 antibody formulation was measured as described in Example 1. These data
are shown in Fig. 1.
Both MgCl2 and CaCl2 at concentrations ranging from about 5 mM to about 20
mM significantly reduced the viscosity of the MYO-029 antibody formulation. NaCl and
NaH2PO4, on the other hand, had little effect in this range.

Thus, calcium chloride and magnesium chloride, at concentrations of about 5 mM
to about 20 mM, are effective viscosity reducing agents for MYO-029 antibody
formulations, unlike sodium chloride and sodium biphosphate.
Example 3
Effect of Calcium Chloride on the Viscosity of a J695 Antibody Formulation
The viscosity of a J695 antibody formulation is measured at two different J695
antibody concentrations, i.e., 100 mg/ml and 300 mg/ml.
The viscosity of the J695 antibody formulation at the higher concentration will be
higher than the viscosity of the 3695 antibody formulation at the lower concentration.
Calcium chloride is added to a final concentration of about 5 mM to 20 mM to the
300 mg/ml J695 antibody formulation. In this case the viscosity of the antibody
formulation is expected to decrease compared to the J695 formulation without calcium
chloride.
Accordingly, calcium chloride, at concentrations of about 5 mM to about 20 mM,
is effective as a viscosity reducing agent for J695 antibody formulations.
Example 4
Effect of Calcium Chloride on the Viscosity of a MYO-028 Antibody Formulation
MYO-028, another anti-myostatin antibody, was concentrated using Centricon
Ultrafree®-4 to a concentration of 95 mg/mL, Calcium chloride was added to MYO-028
according to Table 2 below:

MYO-028 was formulated at 95 mg/mL in 10 mM histidine, 5% sucrose, pH 6.5.
The CaCl2 solution consisted of 10mM histidine, 2% sucrose, 2M CaCl2. The buffer
solution consisted of 10 mM histidine, 5% sucrose, pH 6.5.

The viscosity of these MYO-028 antibody formulations was measured using the
same rheometer method as described in Example 1 with the additional use of a solvent
trap to prevent evaporation, a 100 L liquid sample load of MYO-028 on the plate, and
the test was performed at room temperature.
The data from these experiments are shown in Fig. 2.
The addition of CaCl2 decreased the viscosity of a MYO-028 antibody
formulation at 25 mM and 50 mM CaCl2 compared to a MYO-028 formulation lacking
CaCl2. These data demonstrate the suitability of CaCl2 for use as an agent to reduce
viscosity of a protein formulation, e.g., to formulate a reduced viscosity antibody
formulation.
Example 5
Effect of Calcium Chloride on the Viscosity of a IMA-638 Antibody Formulation
To test the effect of calcium chloride on the viscosity of an IMA-638 antibody
formulation, different amounts of calcium chloride were added with a pipette to aliquots
of the IL-13 antibody, IMA-638. The IMA-638 antibody aliquots had a protein
concentration of approximately 150 mg/mL. Fig. 3 provides a graphical depiction of the
effect of calcium chloride on the viscosity of IMA-638 protein formulations.
The viscosity of the IMA-638 did not show the same reductionin viscosity as
observed for MYO-029. These data demonstrate a method of identifying a suitable
viscosity reducing agent for use with a protein formulation.
Example 6
Effect of Calcium Chloride on the Stability of MYO-029 Antibody
Addition of a compound (i.e., a viscosity reducing agent, e.g., CaCl2) to a protein
formulation could potentially affect the molecules' stability towards freeze-thaw-induced
stresses. This effect could either be detrimental, beneficial, or have no effect on a
proteins' stability during freezing and thawing.
To evaluate the effect of an agent (i.e., CaCl2) on the freeze-thaw-induced
degradation of MYO-029 antibody, the molecule was subjected to 10 freeze thaw cycles
at -80oC and 37°C, in the presence or absence of 5 mM CaCl2. MYO-029 drug substance
was formulated into 10 mM histidine, 2% sucrose, in the presence or absence of calcium

chloride by ultrafiltration and diafiltration. The final protein concentration was
approximately 75 mg/mL. Twenty microliter aliquots were frozen at -80°C and thawed
at room temperature. This was repeated for 5 and 10 iieeze-thaw cycles. Samples were
diluted 25-fold with formulation buffer and analyzed by measuring absorbance at 280 nm
for protein concentration and SEC-HP LC for the percentage of high molecular weight
products (%HMW).
The effect of freeze-thaw-induced degradation was assessed by (i) protein
recovery (absorbance at 280 nm), and (ii) percentage of high molecular weight (% HMW)
formation as determined by size exclusion-high performance liquid chromatography
(SEC-HPLC). HMW formation is the most common degradation pathway for this
molecule. The results of these studies are shown in Fig. 4A and Fig. 4B.
Compared to the corresponding control sample without CaCl2, addition of 5 mM
CaCl2 to the formulation did not have any effect on protein recovery or % HMW
formation. Thus, the addition of calcium chloride does not appear to impact the stability
of MYO-029 antibody formulations. This indicates that suitability of CaCl2 for use as a
viscosity reducing agent in a protein formulation, e.g., in a reduced viscosity antibody
formulation.
Example 7
Effect of Calcium Chloride on the Stability of a MYO-029 Antibody Formulation
Addition of CaCl2 a protein formulation could potentially affect the molecules'
liquid stability over time. This effect could either be detrimental, beneficial, or have no
effect on the proteins' stability during storage.
To evaluate the effect of this agent on the liquid stability of MYO-029 on heat-
induced degradation, formulations containing MYO-029 were subjected to storage at
50°C for up to seven days. Aliquots were taken at various time points and analyzed for
protein concentration by absorbance at 280 nm and % HMW was analyzed by SEC-
HPLC. The data are shown in Fig. 5A and Fig. 5B.
Compared to the control sample, addition of CaCl2 the formulation had no
negative effect on the stability of the protein in the liquid state stored at 50° C. The
percentage of HMW in the drug substance also appeared to be slightly less in the material
containing CaCl2. These data further demonstrate the suitability of using CaCl2 as a

viscosity reducing agent. They also demonstrate a method of determining the suitability
of an agent that reduces viscosity of a protein formulation, e.g., with respect to whether
the agent has an effect on stability of protein in the formulation.
Example 8
Effect of Calcium Chloride on the Stability of Lvophilized MYO-029
Addition of an agent such as CaCl2 to a protein formulation could potentially
affect the proteins' lyophilized dosage forms stability over time. This effect could be
either detrimental, beneficial or have no effect on the proteins' stability during storage.
To evaluate the effect of this excipient on the stability of lyophilized MYO-029, a
formulation containing the molecule is lyophilized both with and without (control) 5 mM
CaCl2 and is subject to storage at 50°C and 4°C for four weeks. Vials are pulled weekly
and analyzed for protein concentration by absorbance at 280 nm, percentage of HMW by
SEC-HPLC, and charge distribution by cation exchange-high performance liquid
chromatography (CEX-HPLC). Vial withdrawal volume and viscosity (one time-point
only) are also measured.
A. Viscosity of Reconstituted Drug Product
The viscosity of the MYO-029 drug product (which is measured in substantially
the same manner as in Example 1) at approximately 150 mg/mL is reduced when 5 mM
calcium chloride is present in the formulation.
B. Withdrawal Volume From the Vial
Trie amount of drug product that can be removed from the vial with a 1 mL
syringe and 21 G needle is improved when CaCl2 is present in the formulation.
C. Protein Concentration
Compared to the control, addition of CaCl2 to the formulation does not affect
protein recovery.

D. High Molecular Weight
Five mM CaCl2 will not have any significant effect on the percentage of HMW
species that is formed after four weeks of storage at 4°C. However, at 50°C, the rate of
HMW formation is expected to be significantly reduced compared to the control.
E. Charge Distribution
The stability time-points are analyzed by CEX-HPLC, a chromatographic tool
used to study charge differences in proteins. In CEX-HPLC, the more negatively charged
molecules elute earlier than the more positively charged molecules. This method is used
to detect deamidation of asparagines residues to either aspartic or iso-aspartic acid.
Deamidation results in an increase in the proteins' net negative charge, and it will elute
earlier from the HPLC column. In this experiment, the effect of calcium chloride has on
protein degradation resulting in a charge change different from that of the control is
investigated. Compared to the control without calcium chloride, the same charge changes
are expected to occur over time. Thus, CaCl2 is expected to have no effect on the charge
distribution of MYO-029 at both storage temperatures.
In summary, compared to a control sample, addition of CaCl2 to a formulation will
have no significant negative effect on the stability of the protein in the formulation
relative to the no calcium chloride experimental control in the lyophilized state when
stored at 4°C and 50°C. In some instances, CaCl2 is found to be beneficial to the stability
of the protein.
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction
with the detailed description thereof, the foregoing description is intended to illustrate and
not limit the scope of the invention, which is defined by the scope of the appended claims.
Other aspects, advantages, and modifications are within the scope of the following
claims.

WHAT IS CLAIMED IS:
1. A method for reducing the viscosity of a protein formulation, the method
comprising;
(a) providing a protein formulation; and
(b) adding calcium chloride or magnesium chloride to the protein formulation to a
concentration of between about 0.5 mM and about 20 mM, wherein the viscosity of the
protein formulation with the calcium chloride or magnesium chloride is reduced
compared to the viscosity of a protein formulation without the viscosity reducing agent.

2. The method of claim 1, wherein the protein is selected from the group consisting
of an antibody, an Ig fusion protein, a receptor, a transcription factor, an enzyme, a
ligand, and a biologically active fragment thereof.
3. The method of claim 1, wherein the protein is an antibody or a biologically active
fragment thereof.
4. The method of claim 1, wherein the protein is an Ig fusion protein.
5. The method of claim 1, wherein the protein is an antibody and the antibody is an
anti-myostatin antibody, an anti-IL-12 antibody, or an anti-IL-13 antibody.
6. The method of claim 5, wherein the anu-tnycstatitt antibody is MYO-O29s
wherein the anti-IL-12 antibody is 3695, and wherein the anti-IL-13 antibody is IMA-638.
7. The method of claim 1, wherein the viscosity of the protein formulation is reduced
by at least about 5% compared to the viscosity of the formulation in the absence of the
viscosity reducing agent.
8. A reduced viscosity formulation, comprising:
(i) a protein;

(ii) a viscosity reducing agent at a concentration of between about 5 mM
and sabout 20 mM in the formulation, wherein the viscosity reducing agent is not
sodium chloride or sodium biphosphate; and
(iii) a buffer;
wherein the pH of the formulation is about 5.5-6.5.
9. The reduced viscosity formulation of claim 8, wherein the protein is selected from
the group consisting of an antibody,, an Ig fusion protein, a receptor, a transcription factor,
an enzyme, a ligand, and biologically active fragments thereof.
10. The method of claim 8, wherein the protein is an antibody or a biologically active
fragment thereof.
11. The method of claim 8, wherein the protein is an Ig fusion protein.
12. The protein formulation of claim 8, wherein the viscosity reducing agent is
calcium chloride, or magnesium chloride.
13. An anti-myostatin antibody formulation, comprising:
(i) an anti-myostatin antibody or a myostatin binding fragment thereof;
(ii) a viscosity reducing agent; and
(iii) a buffer,
wherein the pH of the formulation is about 5.5-6.5.
14. The method of claim 13, wherein the anti-myostatin antibody is a monoclonal
antibody.
15. The method of claim 14, wherein the anti-myostatin antibody is a humanized
monoclonal antibody.
16. The method of claim 14, wherein the anti-myostatin antibody binds myostatin
with a Kd of about 6 X 10-11 M as determined by Biacore™.

17. The method of claim 14, wherein the anti-myostatin antibody is selected from the
group consisting of MYO-022, MYO-028, and MYO-029.
18. The method of claim 13, wherein the anti-myostatin in the formulation is at a
concentration of about 25 mg/ml to about 400 mg/ml.
19. The method of claim 13, wherein the viscosity reducing agent is calcium chloride
or magnesium chloride.
20. The method of claim 13, wherein the viscosity reducing agent is calcium chloride
at a concentration of about 5 mM to about 20 mM.
21. The method of claim 13, wherein the buffer is histidine buffer at a concentration
in the formulation of about 4 mM to about 60 mM.
22. The method of claim 13, wherein the formulation further comprises a
cryoprotectant.
23. The method of claim 22, wherein the cryoprotectant is sucrose or trehalose at a
concentration in the formulation of about 0.5% to about 5% (weight/volume).
24. The method of claim 13, wherein the formulation further comprises a surfactant at
a concentration in the formulation of about 0% to 0.2% (weight/volume).
25. The method of claim 24, wherein the surfactant is polysorbate-20 or polysorbate-
80.
26. The method of claim 13, wherein the formulation further comprises an anti-
oxidant.

27. The method of claim 26, wherein the anti-oxidant is methionine, and the
concentration of methionine in the formulation is between about 2 mM and about 20 mM.
28. The anti-myostatin antibody formulation of claim 13, wherein
(i) the anti-myostatin antibody is a fully humanized anti-myostatin
antibody at a concentration of about 20 mg/ml to about 400 mg/ml;
(ii) the viscosity reducing agent is calcium chloride or magnesium chloride
at a concentration of about 5 rnM to 20 mM; and
(iii) the buffer is a histidine buffer at a concentration of about 5 mM to
about 20 mM,
wherein the pH of the formulation is about 6.0.
29. The formulation of claim 13, wherein the formulation is lyophilized.
30. The formulation of claim 13, wherein the viscosity of the formulation is reduced
by at least about 5% compared to a formulation lacking the viscosity reducing agent.
31. A pharmaceutical composition for the treatment of a disorder selected from the
group consisting of muscular dystrophy, sarcopenia, cachexia, and Type II diabetes,
wherein the pharmaceutical composition comprises an anti-myostatin antibody
formulation of claim 9.
32. A method of treating a disorder selected from the group consisting of muscular
dystrophy, sarcopenia, cachexia, and Type II diabetes, the method comprising
administering a pharmaceutically effective amount of an antibody formulation
. comprising:
(i) an anti-myostatia antibody or a myostatin binding fragment thereof;
(ii) a viscosity reducing agent; and
(iii) a buffer,
wherein the pH of the formulation is about 5.5-6.5.

33. The method of claim 32, wherein the antibody formulation is administered by
injection, intravenous infusion, or pulmonary administration via a nebulizer or as a dry
powder.
34. A kit comprising a container containing the formulation of claim 1.
35. The kit of claim 34, further comprising directions for administration of said
formulation.
36. A method for reducing the viscosity of a protein formulation, the method
comprising
(i) providing a protein forumlation; and
(ii) adding a viscosity reducing agent to the protein formulation, wherein
the viscosity reducing agent is calcium chloride or magnesium chloride,
and wherein the concentration of the viscosity reducing agent in the
formulation is between about 1 mM and about 25 mM.
37. A method for identifying a reduced viscosity protein formulation, the method
comprising
(i) providing a protein formulation;
(ii) adding a viscosity reducing agent to the protein formulation, wherein
the viscosity reducing agent is calcium chloride or magnesium chloride, and
wherein the concentration of the viscosity reducing agent in the formulation is
between about 1 mM and about 25 mM, thereby forming a potential reduced
viscosity formulation; and
(iii) determining the viscosity of the potential reduced viscosity protein
formulation,
wherein when the viscosity of the potential reduced viscosity protein formulation
is reduced compared to the viscosity of the protein formulation without the viscosity
reducing agent, the formulation is a reduced viscosity formulation.

38. The method of claim 1, wherein the method further comprises determining the
stability of the protein formulation.
39. The method of claim 37, wherein the method further comprises determining the
stability of the protein formulation.
40. The method of claim 39, wherein the stability of the protein formulation is
determined after freeze-thawing the protein formulation, after storage of the protein
formulation at 50°C for 1-7 days, or after lyophilization.
41. The method of claim 39, wherein stability is determined by assaying the
percentage of high molecular weight species, the percentage of low molecular weight
species, or the charge distribution of the protein formulation compared to a control.

Protein formulations and methods for reducing the viscosity of a protein formulation are provided. The method for
reducing the viscosity of a protein formulation comprises adding a viscosity reducing agent, such as calcium chloride or magnesium
chloride to the protein formulation.