METHODS AND COMPOSITIONS WITH REDUCED OPALESCENCE
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Application Serial No. 60/851,651, filed
on October 12, 2006, the content of which is hereby incorporated by reference in its
entirety.
TECHNICAL FIELD
The invention relates to methods for reducing the opalescence of protein
preparations, as well as compositions, e.g., pharmaceutical compositions, of concentrated
protein preparations having decreased opalescence.
BACKGROUND
Proteins, including antibodies, have been used in drug therapy for the past twenty
years. In order to achieve high therapeutic doses, antibodies are typically formulated at
high concentrations (about 10 mg/ml to 100 mg/ml or greater) (Brekke, O.H. and Sandlie,
I. (2003) Nat. Rev. Drug Discov. 2:52-62). Certain modes of protein administration
typically require highly concentrated protein formulations. For example, subcutaneous
administration of therapeutic antibodies are often formulated at concentrations greater
than about 100 mg/ml. Some of these concentrated protein formulations develop an
opalescent appearance at high concentrations, a property often referred to as opalescence
(Schellekens, H. (2002) Nat. Rev. Drug Discov. 1:457-462).
An opalescent appearance in a concentrated protein solution may result from a
variety of factors, including protein concentration, its effect in Rayleigh scatter,
temperature, the nature of solute-solute interactions, among others. When a protein is
susceptible to opalescence, the opalescent appearance usually increases as the protein
concentration increases. The similarity of opalescent solutions to aggregated protein
solutions has raised concerns with respect to its loss of protein activity and potential to
cause immunogenicity in pharmaceutical formulations (Pinckard et al. (1967) Clin Exp.
Immunol. 2:331 -340; Robbins et al. (1987) Diabetes 36:838-845; Cleland et al. (1993)
Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377).

Thus, there is a need for developing formulations of highly concentrated
therapeutic proteins, e.g., therapeutic antibodies, having reduced opalescence.
SUMMARY
The invention is based, at least in part, on the discovery that reducing the
concentration of salt, e.g., sodium chloride, in an antibody preparation reduces the
opalescent appearance of the preparation and/or the formation of high molecular weight
species in the preparation. Thus, methods of reducing the opalescent appearance of
protein, e.g., antibody, preparations, e.g., by modifying the ionic strength of the
preparations, as well as compositions, e.g., pharmaceutical compositions, of concentrated
proteins having decreased opalescence are disclosed. Purification methods which
monitor and/or reduce the salt concentration(s) at selected steps in the purification process
are also disclosed.
Accordingly, in one aspect, the invention features a method for decreasing the
opalescence of, or reducing the amount of high molecular weight species in, a protein
preparation, e.g., an antibody preparation (e.g., a solution that includes a protein, e.g., an
antibody). The method includes modifying, e.g., decreasing or increasing, the ionic
strength of the preparation such that the opalescent appearance of, and/or the amount of
high molecular weight species in, the protein preparation is reduced and/or eliminated. In
embodiments, the ratio of the ionic strength to the protein concentration, e.g., the
antibody concentration, in the protein preparation is modified or altered, e.g., decreased
or increased, such that the opalescent appearance of, and/or the amount of high molecular
weight species in, the protein preparation is reduced and/or eliminated. In embodiments,
the protein preparation shows unwanted opalescence and/or high molecular weight
species prior to reducing the ionic strength (e.g., a turbidity greater than about 1, 2, 3, 4 or
5, or more European Pharmacopeia standard and/or a percentage of high molecular
weight species corresponding to about 2%, 3%, 5%, 10%, 15%, 20%, 30% or higher of
the percent mass of the protein preparation) at a pharmaceutically effective concentration,
e.g., about 5, 10, 25, 50, 75,100, 125, 150 mg/ml for antibody preparations.
In embodiments, the protein in the preparation is a secreted protein, e.g., an
antibody, an antigen-binding fragment of an antibody, a binding domain-immunoglobulin
fusion (e.g., SMIP™), a soluble receptor, a receptor fusion, a cytokine, a growth factor,
an enzyme, or a clotting factor, as described in more detail herein below. In embodiments
where the protein is an antibody, it can include at least one, and preferably two full-length

heavy chains, and at least one, and preferably two light chains. The term "antibody" as
used herein includes an antibody fragment or a variant molecule such as an antigen-
binding fragment {e.g., an Fab, F(ab')2, Fv, a single chain Fv fragment, and a heavy chain
fragment (e.g., a camelid VHH). The antibody can be a monoclonal or single-specificity
antibody. The antibody can also be a human, humanized, chimeric, CDR-grafted, or in
vitro generated antibody.In yet other embodiments, the antibody has a heavy chain
constant region chosen from, e.g., IgGl, IgG2, IgG3, or IgG4. In another embodiment,
the antibody has a light chain chosen from, e.g., kappa or lambda. In one embodiment,
the constant region is altered, e.g., mutated, to modify the properties of the antibody (e.g.,
to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the
number of cysteine residues, effector cell function, or complement function). Typically,
the antibody specifically binds to a predetermined antigen, e.g., an antigen associated
with a disorder, e.g., a neurodegenerative, metabolic, inflammatory, autoimmune and/or a
malignant disorder. Exemplary antibodies that can be used in the methods of the
invention include, but are not limited to, antibodies against an Aβ peptide, interleukin-12
(IL-12), interleukin-13 (IL-13), interleukin-22 (IL-22), 5T4, and growth and
differentiation factor-8 (GDF-8). In embodiments, the antibody is an anti-GDF-8
antibody, e.g., Myo-029.
In embodiments, the protein self-aggregates at a higher protein and/or salt
concentration, e.g., has a negative second virial coefficient, e.g., in a high salt preparation
(e.g., a preparation having a salt concentration of about 50 to 200 mM (e.g., about 100 to
150 mM of a salt)). For example, the protein has a second virial coefficient of about -1 x
10"' to -l,about-1xl0-2to-10x 10-2, about-1 x10-3to-10x 10-3, about-1 x 10-4to-10
x 10-4 , about -i x 10 -5 to -10 x 10" mol-mg/g2 in a preparation containing, e.g., 150 mM
sodium chloride. In other embodiments, the protein has a positive virial coefficient, e.g.,
about 1 x 10-5 to 10 x 10-5, about 1 x 10-4 to 10 x 10-4, about 1 x 10-3to 10 x 10-3, about 1
x 10-2 to 10 x 10-2 mol-mg/g2 in a preparation containing, e.g., 150 mM sodium chloride.
In embodiments where the protein is an antibody or a fragment thereof, the antibody is
present at a concentration of about 0.1 to about 1,000 mg/ml, typically about 0.5 to about
500, about 1 to 400, about 5 to 300, about 10 to 250, about 15 to 200, about 20 to 150,
about 50 to 100 mg/ml prior to, and/or after practicing the methods of the invention.
In embodiments, the ionic strength of the protein preparation is modified, e.g.,
reduced, by decreasing the concentration of a salt present in the preparation, and/or by
replacing the salt used in the preparation with a salt that induces less opalescence. The

salt in the preparation can be chosen from one or more of, e.g., sodium chloride, calcium
chloride, magnesium chloride or sodium phosphate. The salt concentration in the
preparation can be reduced to a lower concentration, e.g., a concentration at least about
two-, three-, five-, ten- or one hundred-fold lower than the concentration in the opalescent
preparation. For example, the salt {e.g., sodium chloride) concentration in the protein
preparation can be reduced to less than about. 300 mM, typically, less than about about 200,
150, 100, 75, 50,40, 30, 25, 20,15, 10, 5,4, 3, 2, 1 or 0 mM. The salt concentration in
the protein preparation can be modified, e.g., reduced by, e.g., applying one or more
filtration methods chosen from one or more of: ultra-filtration or dialysis. Alternatively
or in addition, the ionic strength of the protein preparation is modified, e.g., decreased or
increased, by replacing one or more first salt(s), e.g., a high opalescent inducer, with one
or more second salt(s), e.g., a lower opalescent inducer. The following exemplary salts
were ranked according to the level of opalescence induction in antibody preparations, in
the following order from high to low opalescence inducer: sodium chloride (NaCl),
sodium phosphate (NaHPO4, magnesium chloride (MgCl2) and calcium chloride (CaCl2).
Accordingly, in embodiments, the first salt is NaCl, and is replaced with a second salt
chosen from one or more of, e.g., NaHPO4, MgCl2, or CaCl2; the first salt is NaHPO4, and
is replaced with a second salt chosen from one or more of, e.g., MgCl2 or CaCl2; the first
salt is MgCl2, and is replaced with a second salt, CaCl2. In embodiments, the first salt is
replaced by the second salt in the protein preparation by removing the first salt, e.g., by
dialysis or ultrafiltration, and adding the second salt to the protein preparation. In
embodiments, the concentration of the second salt is greater than the concentration of the
first salt. In embodiments, the concentration of the second salt is equal to the
concentration of the first salt In other embodiments, the concentration of the second salt
is less than the concentration of the first salt.
In other embodiments, the ionic strength of the protein preparation is modified,
e.g., decreased or increased, in the process of making and/or purifying the protein, e.g., by
decreasing the concentration of a high opalescent inducer salt used. The embodiments
include: (i) (optionally) evaluating whether the protein preparation forms an opalescent
solution at one or more protein and/or salt concentrations; (ii) (optionally) providing the
salt concentration in the protein preparation; and/or (iii) modifying, e.g., reducing, the
ionic strength of the protein preparation (e.g., modifying the ratio of the ionic strength to
the protein concentration in the protein preparation). Steps (ii) and/or (iii) can be
repeated and/or carried out in any order, as needed. Step (i) can be performed by

detecting the opalescence and/or presence of high molecular weight species in one or
more samples containing different concentrations of the protein and/or salt, for example,
as described in the Examples herein. The ionic strength of the protein preparation can be
modified, e.g., reduced, e.g., by applying one or more filtration methods described herein.
Alternatively or in addition, the ionic strength of the protein preparation can be decreased
by reduced the ionic strength, e.g., the salt concentration, of one or more steps in the
process of making and/or purifying the protein. Such reduction in ionic strength in the
process of making and/or purifying the protein can be effected by decreasing the
concentration of a salt used in the one or more steps. In other embodiments, the ionic
strength is modified by replacing the salt used in the one or more steps with a lower
opalescent inducer. In certain embodiments, the protein in the preparation is made
recombinantly, e.g., expressed using recombinant techniques as, e.g., a cell-bound, a
soluble or a secreted protein. In such embodiments, the protein is expressed and
separated from the recombinant host (e.g., secreted into the medium and separated, by,
e.g., centrifugation or filtration). The separated protein is further purified by methods
known in the art, including, but not limited to, Protein A chromatography, affinity
chromatography, hydrophobic interaction chromatography, immobilized metal affinity
chromatography, size exclusion chromatography, diafiltration, ultrafiltration, viral
removal filtration, anion exchange chromatography, hydroxyapatite chromatography
and/or cation exchange chromatography. The protein may be further lyophilized and/or
reconstituted in a buffer solution. The methods of the invention include modifying, e.g.,
reducing, the ionic strength, e.g., the salt concentration, of the solutions (e.g., washing,
load, elution, reconstitution solutions), used in one or more of the aforesaid steps.
Without being pound by theory, it is believed that residual amounts of salt can be
present in protein preparations, even after applying filtration methods such as ultra-
filtration and dialysis. Such residual salt amounts are characterized in the art as the
"Donnan effect." (Miro, M et al. (2004) Analytica Chimica Acta 512(2):311-317). It is
believed that such residual salt amounts might lead to protein preparations having an
opalescent appearance, particularly when the protein in the preparation has a tendency to
self-aggregate, e.g., it has a negative virial coefficient. Thus, modifying the ionic
concentration using the methods disclosed herein can have broad applicability to protein
purification techniques.
In embodiments, the reduction in opalescence of the protein preparation is
detected by evaluating the turbidity of the solution. For example, the turbidity of the

protein preparation is reduced to about 5, 4, 3, 2, 1 or less European Pharmacopeia
standard.
In other embodiments, the reduction in opalescence of the protein preparation is
determined by evaluating a change in the second virial coefficient of the protein. For
example, a two-, three-, five-, ten- or one hundred-fold change, e.g., an increase or a
decrease in the second virial coefficient of the protein is detected after modifying, e.g.,
reducing, the ionic strength of the protein preparation. For example, the virial coefficient
changes, e.g., decreases, from anywhere between about 1 x 10-2 and 1 x 10-3 to anywhere
between about 1 x 10-3 and 1 x 10-4
In yet other embodiments, the reduction in opalescence in the protein preparation
is detected by determining the amount of high molecular weight species. The high
molecular weight species typically have a molecular weight of about 104, more typically,
about 105, 106, 107, 108, 109, 1010, 10", 1012, 1013, or higher. The weight average
molecular weight of the protein species in the preparation can be detected using one or
more of: light scattering techniques, e.g., static and dynamic light scattering, asymmetric
flow field flow fractionation, or SEC-HPLC, as, for example, described in the Examples
below. In embodiments, a two-, three-, four-, five-, six-, seven-, eight-, nine-, ten-, fifty-
or one hundred-fold decrease in high molecular weight species is indicative of a reduction
of opalescence in the protein preparation. For example, the percentage mass of the high
molecular weight species decreased from about 30 to 40% of the total protein mass to less
than about 5%, with a concomitant increase in the percentage mass of the non-aggregated
protein from about 60 to 70% of the total protein mass to about 95% or higher (as
detected by dynamic light scattering).
In another aspect, tne invention features a method of decreasing the formation,
and/or reducing the amount, of high molecular weight species in a protein, e.g., antibody,
preparation (e.g., a protein, e.g., antibody, preparation as described herein). The method
includes modifying, e.g., decreasing or increasing, the ionic strength of the preparation
(e.g., modifying the ratio of the ionic strength to the protein concentration in the protein
preparation), such that the amount of high molecular weight species in the protein
preparation is reduced and/or eliminated.
In embodiments, the high molecular weight species are protein aggregates, which
can be substantially reversibly dissociated upon reducing the protein and/or salt
concentration. The high molecular weight species typically have a molecular weight of
about 104kDa, more typically, about 105,106,107,108,109,1010,10", 1012,10l3kDa, or

higher. The weight average molecular weight of the protein species in the preparation
can be detected using one or more of: light scattering techniques, e.g., static and dynamic
light scattering, asymmetric flow field flow fractionation, or SEC-HPLC, as, for example,
described in the Examples below. In embodiments, a two-, three-, four-, five-, six-,
seven-, eight-, nine-, ten-, fifty- or one hundred-fold decrease in high molecular weight
species is indicative of a reduction of opalescence in tne protein preparation, for
example, the percentage mass of the higher molecular weight species is decreased from
about 30 to 40% of the total protein mass to less than about 5%, with a concomitant
increase in the percentage mass of the non-aggregated protein from about 60 to 70% of
the total protein mass to about 95% or higher (as detected by dynamic light scattering).
In embodiments, the decrease in ionic strength of the preparation is effected by the
methods disclosed herein.
In another aspect, the invention features a method of improving the efficiency of a
production and/or purification process of a protein, e.g., antibody, preparation (e.g., a
protein, e.g., antibody, preparation as described herein). The method includes: (i)
(optionally) evaluating whether the protein preparation forms an opalescent solution at
one or more protein and/or salt concentrations; (ii) modifying, e.g., decreasing, the ionic
strength of the preparation, such that the amount of high molecular weight species in the
protein preparation is reduced and/or eliminated. Step (i) can be performed by detecting
the opalescence and/or presence of high molecular weight species in one or more samples
containing different concentrations of the protein and/or salt, for example, as described in
the Examples herein. The ionic strength of the protein preparation can be modified, e.g.,
reduced, e.g., by applying one or more filtration methods described herein; replacing the
salt used in the protein preparation with a lower opalescent inducer, e.g., as described
herein; and/or reducing the ionic strength of one or more steps in the process of making
and/or purifying the protein, e.g., as described herein.
In yet another aspect, the invention features a method of improving the production
of an antibody, e.g., selecting a salt or a salt concentration for use in a process of making
an antibody. The method includes (i) recovering an antibody solution (e.g., eluting an
antibody from a matrix; recovering a dialyzed solution or other filtrate; and/or
solubilizing a dry preparation, e.g., a Iyophilized or dried preparation with a first solution
having a first ionic strength, e.g., first salt concentration); (ii) evaluating the level of
opalescence and/or presence of high molecular weight species in the antibody solution;

wherein (a) if the level of opalescence and/or high molecular weight species is equal to or
less than a predetermined level, then selecting said antibody solution for use in the
antibody product, e.g., a pharmaceutical composition that includes the antibody solution;
or (b) if the level of opalescence and/or high molecular weight species is greater than a
predetermined level, then selecting a second solution having a second, e.g., lower, ionic
strength (e.g., a second,e.g., lower, salt concentration). The level of opalescence and/or
presence of high molecular weight species in the second solution containing the antibody
can be evaluated, as needed, and additional solutions can be added until the level of
opalescence reaches (or is less than) the predetermined level.
In embodiments, the antibody solution is recovered from one or more protein
purification methods known in the art, including, but not limited to, Protein A
chromatography, affinity chromatography, hydrophobic interaction chromatography,
immobilized metal affinity chromatography, size exclusion chromatography, diafiltration,
ultrafiltration, viral removal filtration, anion exchange chromatography, hydroxyapatite
chromatography and/or cation exchange chromatography. The protein may be further
lyophilized and/or reconstituted in a buffer solution.
In embodiments, the predetermined level of the antibody (first, second and
additional) solution is a turbidity value of about greater than about 1, 2, 3, 4, 5 or more
European Pharmacopeia standard, typically about 3, and/or an increase in high molecular
weight species to 1%, 2%, 3%, 5%, 10%, 15%, 20%, 30% or higher of the percent mass
of the protein preparation, typically about 2% at a pharmaceutical ly effective
concentration, e.g., about 5, 10, 25, 50, 75, 100, 125, or 150 mg/ml for antibody
preparations.
In another aspect, the invention features an antibody purification method or an
antibody preparation, wherein the amount of residual salt concentration bound to the
antibody is less than that seen after the antibody has been exhaustively dialyzed, e.g.,
dialyzed in 0 mM salt.
In another aspect, the invention features a method of determining the propensity
of a protein, e.g., an antibody, preparation, as described herein to have an opalescent
appearance. The method includes: (i) (optionally) identifying a protein as having a
tendency to self-aggregate, e.g., by determining the surface protein charge and/or second
virial coefficient; and/or (ii) detecting formation and/or disappearance of high molecular
weight species upon increasing and/or decreasing, respectively, the protein and/or salt
concentration in one or more samples containing the protein in solution. A negative virial

coefficient and/or an increase in high molecular weight species upon increasing salt
and/or protein concentration are indicative of an increase propensity of the protein in the
preparation to have an opalescent appearance.
In yet another aspect, the invention features a method of evaluating the
opalescence of a protein, e.g., an antibody, sample. The method includes: providing a protein e.g., an antibody, sample; determining the turbidity and/or presence of high
molecular weight species at one or more (e.g., at least two, three or more) salt, e.g., NaCl,
concentrations; providing a report on the level of opalescence in the protein, e.g.,
antibody, sample as a function of turbidity and/or presence of high molecular weight
species at the one or more salt, e.g., NaCl, concentrations.
In other aspects, protein, e.g., antibody, preparations (as well as pharmaceutical
compositions and/or formulations that include the protein preparations disclosed herein)
having reduced opalescence are also within the scope of the invention. In embodiments,
the protein preparations are produced by the methods described herein.
In other embodiments, the protein preparation includes at least 70%, 80%, 90%,
95%, 96%, 97%, 98%, 99% or higher percentage mass of non-aggregated species
compared to the high molecular weight species (as detected, e.g., by dynamic light
scattering) prior to removal of one or more high molecular weight species, e.g., a
filtration or centrifugation step. In embodiments, a modification, e.g., a decrease, in the
ionic strength of the protein preparation causes an increase of two-, three-, four-, five-,
six-, seven-, eight-, nine-, ten-, fifty- or one hundred-fold in the percentage of non-
aggregated protein compared to high molecular weight species.
In yet another aspect, the invention features a pharmaceutically acceptable
antibody preparation that incrudes an antibody, e.g., a human or a humanized antibody, at
a concentration of at least about 50, 75, 100,125, or 150 mg/ml in less than about 50, 40,
30, 20, 10, 5, or 1 mM salt, e.g., NaCl.
In another aspect, the invention features a method of providing an antibody
preparation that includes: (i) providing an antibody solution, e.g., of a human or
humanized antibody having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or
higher percentage mass of non-aggregated species prior to removal of one or more high
molecular weight species, e.g., a filtration or centrifugation step; (ii) performing one or
more purification steps to provide an intermediate antibody solution; and (iii) processing
the intermediate solution to provide an antibody preparation having a concentration of at

least about 50 to 150 mg/ml and a turbidity of less than about 5, 4,3,2, 1 or less
European Pharmacopeia standard.
Other aspects, features and advantages will be apparent from the description of the
preferred implementations thereof and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows images of Antibody Ml (also referred to herein as "Myo-029")
solutions exposed to increasing NaCl concentration (showing a visually perceptible
opalescence increase).
Figure 2 shows dynamic light scattering plots for Antibody Ml solutions in 0 mM
NaCl and 100 mMNaCl.
Figure 3 shows an asymmetric flow field flow fractionation plot for an Antibody
Ml solution in 100 mM NaCl.
Figure 4 shows a SEC-HPLC chromatograph for an Antibody Ml solution in 100
mMNaCl.
Figure 5 shows images of Antibody Ml solutions that have been dialyzed from 0
mM NaCl to 100 mM NaCl and back to 0 mM NaCl (showing visibly reversible
opalescence with changes in salt concentration).
Figure 6 shows dynamic light scattering plots of Antibody Ml solutions showing
reversible formation of opalescent species: (a) 100 mM NaCl, which was dialyzed from
OmM NaCl, and (b) 0 mM NaCl, which was dialyzed from 100 mM NaCl.
Figure 7 shows images of Antibody M2 solutions with increasing NaCl
concentration (showing no visually perceptible opalescence increase).
Figure 8 shows dynamic light scattering plots for Antibody M2 solutions in 0 mM
NaCl and 150mMNaCl.
Figure 9 shows dynamic light scattering plots for Antibody M3 solutions in 0 mM
NaCl and 150mMNaCl.
Figure 10 shows a second virial coefficient plot for Antibody Ml at 100 mM
NaCl.
Figure 11 shows a second virial coefficient plot for Antibody M2 at 100 mM
NaCl.
Figure 12 shows images of Antibody Ml solutions containing different salts
showing opalescence after one hour.

Figure 13 shows images of Antibody Ml solutions containing different salts
showing opalescence after two weeks.
Figure 14 shows a plot of OD400 nm versus number of temperatures cycles for
Antibody Ml solutions containing different salts.
Figure 15 shows a plot of percent high molecular weight versus salt concentration
for Antibody M1 solutions containing different salts.
Figure 16 shows a plot of OD400 nm versus salt concentration for Antibody Ml
solutions containing different salts.
Figure 17 shows an expanded region of the plot shown in Figure 16.
Figure 18 shows a plot of OD400 nm versus Antibody Ml concentration in 20
mM CaCl2.
DETAILED DESCRIPTION
The present application discloses methods of reducing the opalescent appearance
of a protein preparation by modifying the ionic strength of the preparation, as well as
compositions, e.g., pharmaceutical compositions, of concentrated protein with decreased
opalescence are disclosed. Purification methods which monitor and/or reduce the salt
concentrations at selected steps are also disclosed.
In one embodiment, the impact of salt concentration, i.e., ionic strength, on the
opalescent appearance of concentrated antibody preparations was evaluated for three
antibodies: Antibody Ml (also referred to herein as "Myo-029"), Antibody M2, and
Antibody M3. As described in more detail in the appended Example, it was found that all
three antibodies contain a relatively small amount of an extremely large species in just 10
mM histidine at pH 6.0. This large species was too large to be separated using an SEC-
HPLC column. Increasing the ionic strength by increasing the amount of sodium chloride
in the antibody preparation increases the opalescence, as well as the appearance of a.
liquid-liquid phase separation for Antibody Ml (see e.g., Figures 1-4). The opalescent
appearance is reversible upon salt removal as shown for Antibody Ml in Figures 5-6. A
large species was observed for Antibody Ml using asymmetric Flow Field Flow
Fractionation (aFFFF), which was later confirmed using dynamic light scattering
techniques (see Figure 2). Although Antibodies M2 and M3 did not show a significant
increase in opalescence detectable by visual inspection compared to Antibody Ml (see
e.g., Figure 7), high molecular weight species were detected for both antibodies using
light scattering techniques (shown in Figures 8 and 9).

A negative virial coefficient calculated for Antibody Ml is consistent with the
aggregation/association tendencies of Antibody Ml, as compared to Antibody M3 and
Antibody M2 (both of which have a positive virial coefficient) (compare Figure 10 with
Figure 11).
The results shown herein indicate that opalescent appearance of the antibodies studied is due primarily to reversible self-association, which is further induced by
increasing ionic strength. Accordingly, as disclosed by the present invention, in order to
reduce the opalescent appearance of concentrated antibody preparations (particularly
those having a tendency to aggregate), the ionic strength of the antibody preparation
should be decreased from the formulation and/or limited in the processing method.
In order that the present invention may be more readily understood, certain terms
are first defined. Additional definitions are set forth throughout the detailed description.
The term "opalescence" or "opalescent appearance" refers to the degree of
turbidity detected in a solution, e.g., a protein preparation, as a function of the
concentration of one or more of the components in the solution, e.g., protein and/or salt
concentration. The degree of turbidity can be calculated by reference to a standard curve
generated using suspensions of known turbidity. Reference standards for determining the
degree of turbidity for pharmaceutical compositions can be based on the European
Pharmacopeia criteria (European Pharmacopoeia, Fourth Ed., Directorate for the Quality
of Medicine of the Council of Europe (EDQM), Strasbourg, France). According to the
European Pharmacopeia criteria, a clear solution is defined as one with a turbidity less
than or equal to a reference suspension which has a turbidity of approximately 3
according to European Pharmacopeia standards. Nephelometric turbidity measurements
can dotect Rayleigh scatter, which typically, whicn typically changes linearly with concentration, in the
absence of association or nonideality effects.
For example, ideal solutions behave consistently with the following formula:

where M is molecular weight, K is a constant, Rθ is the Rayleigh ratio (which
combines a number of experimental parameters), and C is concentration. Whereas, non-
ideal solutions can be described using the following formula:


where B is the second virial coefficient. To obtain M, KC/Rθ is extrapolated to
zero angle and zero concentration. A Zimm plot places KC/Rθ on the ordinate and
sin2(0/2) + kC on the abscissa, where k is an arbitrary constant. The Zimm plot, thus,
allows both KC/Re zero angle and zero concentration extrapolations to be made on the
same graph. With the Zimm plot, M and B are respectively the intercept and slope of the
zero angle line.
The term "second virial coefficient" is art-recognized to refer to a measure of the
excluded volume effects and intermolecular interactions (Tessier, P.M. et al. (2003)
Current Opinion in Biotechnology 14(5):512-516). A positive second virial coefficient is
typically indicative of both excluded volume effects and intermolecular repulsive
interactions. In contrast, a negative second virial coefficient typically indicates attractive
intermolecular interactions. The balance of these intra- and intermolecular interactions
determine the sign and amplitude of the second virial coefficient.
The term "ionic strength" refers to the concentration of ions in a solution. If "I" is
ionic strength:

where ci is the molarity concentration of ion i, zi is the charge of that ion, and the
sum is taken over all ions in the solution. (IUPAC Compendium of Chemical
Terminology, 2nd Ed. (1997)).
The term "protein" as used herein refers to one or more polypeptides that can
function as a unit. The term "polypeptide" as used herein refers a sequential chain of
ammo acids linked together via peptide bonds. The term "polypeptide" is used to refer to
an amino acid chain of any length, but one of ordinary skill in the art will understand that
the term is not limited to lengthy chains and can refer to a minimal chain comprising two
amino acids linked together via a peptide bond. If a single polypeptide can function as a
unit, the terms "polypeptide" and "protein" may be used interchangeably. The terms
proteins and polypeptides include antibodies, receptor fusions, SMEP™, growth factors,
cytokines, clotting factors and enzymes, as described in more detail below.
The term "antibody" refers to any immunoglobulin or fragment thereof, and
encompasses any peptide or polypeptide comprising an antigen-binding site. The term
includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, bi-
specific, humanized, de-immunized, human, camelid, rodent, single-chain, chimeric,

synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies. The
term "antibody" also includes antibody fragments and variant molecules such as Fab,
F(ab')2, Fv, scFv, Fd, dAb, VHH, and other antibody fragments and variant molecules that
retain antigen-binding function. Typically, such fragments would comprise an antigen-
binding domain.
The term "protein preparation'' refers to any composition containing at least one
protein, e.g., an antibody, in solution, which is capable of forming high molecular weight
species upon an increase in the ionic strength of the preparation. The protein preparation
may contain the same or different proteins, e.g., antibodies having different binding
specificity.
The term "high molecular weight species" refers to an association of at least two
proteins, e.g., antibodies. In embodiments, the protein association leads to the formation
of higher order aggregates of monomelic protein. The association may arise as a result of
non-covalent (e.g., electrostatic, van der Waals) protein-protein interactions. The
association is typically reversible upon reducing the ionic strength of the protein
preparation. The proteins may be the same or different, e.g., antibodies having different
binding specificity. The high molecular weight species typically have a molecular weight
of about 104, more typically, about 10s, 106, 107, 108, 109, 1010, 10", 1012, 10l\ or higher.
The aggregation of proteins to form high molecular weight species can be
monitored using several analytical techniques, including, but not limited to, optical
density, asymmetric flow field flow fractionation, SEC-HPLC, static light scattering, and
dynamic light scattering, as described in more detail herein.
Turbidity of a protein sample can be measured as optical density (absorbance of
light at a spccific Wavelength). An optical density measurement can be made using
spectrophotometry (e.g., using a SpectraMax UV-Vis at a wavelength of 400nm). For a
more detailed description of how to measure optical density, see e.g., Eckhardt BM
(1994) JPharm Sci Technol. 48(2):64-70.
Asymmetric flow field flow fractionation (AF4) is a type of asymmetric field flow
fractionation. AF4 is a method capable of rapid fractionation and high resolution
characterization of various particles including bio-molecules (Giddings, J. C; Yang, F. J.;
Myers, M. N., Flow-field-flow fractionation: a versatile new separation method, 193
Science 1244-1245 (1976); Giddings, J. C; Yang, F. J.; Myers, M. N., Theoretical and
experimental characterization of flow field-flow fractionation, 48 Anal. Chem. 1126-1132
(1976)). AF4 is capable of separating particles ranging from a few nanometers to a few

micrometers. Field flow fractionation separation occurs in a thin flow channel
(comparable to a chromatographic separation column). An aqueous or organic solvent
carries the sample through this channel. The flow through the channel, the first force
exerted on the sample, is laminar due to the low channel height. A second force is
generated perpendicular to the channel flow. In AF4, one side of the flow channel is a
membrane and the second force is fluid flow across the channel through the membrane.
Particle separation occurs in this system as a result of these two forces. First, the velocity
gradient due to the laminar flow within the channel causes particles in the center of the
channel to move more quickly along the channel and be separated from those closer to the
sides of the channel. Second, the second force forces the sample toward the membrane.
Size separation occurs because the smaller molecules diffuse back toward the center of
the channel more quickly than larger particles and hence are separated from the larger
particles due to the quicker solvent flow toward the center of the channel.
The acronym SEC-HPLC stands for size exclusion chromatography-high
performance liquid chromatography. SEC and HPLC are individually well known
analytical techniques as is the combination SEC-HPLC analytical technique. (Gamick, R.
L., Ross, M. J., Du Mee, C. P., Encyclopedia of Pharmaceutical Technology, Ed. J.
Swarbrick, J.C., Boylan, Vol. 1: 253 (Marcel Dekker, Inc., New York, 1988)). Without
delving into the theories behind each technique, SEC, also know as gel permeation
chromatography, separates molecules based on size due to the molecules' ability to move
through a gel matrix, and HPLC separates molecules based on diffusion coefficients as
the molecules are moved past a stationary medium under high pressure.
Static and dynamic light scattering are two different experimental methods for
measuring the patterns of light scattered from a solvent/solute system. (Berne, B. and
Pecora, R. Dynamic Light Scattering with Applications to Chemistry, Biology and Physics
(Wiley, New York, 1976)). Static and dynamic light scattering give complementary
pieces of information, and for this reason they are commonly used in tandem for
characterization of solutions.
Static light scattering involves measuring the angular dependency of the time-
mean-intensity of laser light scattered by the particles in solution. The size and structure
of the particles impact the light scatter intensity as a function of the detector angle. It has
been well established that in the limit of low scatter vectors and low concentrations, the
angular distribution of the scattered intensity becomes independent of the particle shape.
(Zimm, B., The scattering of light and the radial distribution function of high polymer

solutions, 16 J. Chem.Phys. 1093-9(1948)). As discussed above, by extrapolation of
(Kc/Rθ) to zero angle and zero concentration, several factors including the average
molecular weight, radius of gyration and second virial constant can be estimated.
Dynamic light scattering involves measuring the time-dependent fluctuations in
the intensity of scattered light that occurs because the particles in solution, in this case
"proteins, are undergoing random, Brownian motion. By analyzing these intensity
fluctuations, the distribution of diffusion coefficients of the particles can be determined.
The distribution of diffusion coefficients can then be converted into a size distribution
using well known theories. The upper sized limit for dynamic light scattering is sample
density dependent, i.e., the particles must be able to move, so the upper limit is often the
point at which the sedimentation of particles dominates the diffusion process. The lower
limit will depend on the excess scattered light the sample generates compared to the
suspending medium, as well as other factors including sample concentration, relative
refractive indices, laser power, laser wavelength, detector sensitivity, etc.
Proteins
In certain embodiments, the proteins in the protein preparations are produced
recombinantly. The terms "recombinantly expressed protein" and "recombinant protein"
as used herein refer to a polypeptide expressed from a host cell that has been manipulated
by the hand of man to express that polypeptide. In certain embodiments, the host cell is a
mammalian cell. In certain embodiments, this manipulation may comprise one or more
genetic modifications. For example, the host cells may be genetically modified by the
introduction of one or more heterologous genes encoding the polypeptide to be expressed.
The heterologous recombinantly expressed polypeptide can be identical or similar to
polypeptides that are normally expressed in the host cell. The heterologous
recombinantly expressed polypeptide can also be foreign to the host cell, e.g.,
heterologous to polypeptides normally expressed in the host cell. In certain
embodiments, the heterologous recombinantly expressed polypeptide is chimeric. For
example, portions of a polypeptide may contain amino acid sequences that are identical or
similar to polypeptides normally expressed in the host cell, while other portions contain
amino acid sequences that are foreign to the host cell. Additionally or alternatively, a
polypeptide may contain amino acid sequences from two or more different polypeptides
that are both normally expressed in the host cell. Furthermore, a polypeptide may contain
amino acid sequences from two or more polypeptides that are both foreign to the host

cell. In some embodiments, the host cell is genetically modified by the activation or
upregulation of one or more endogenous genes.
Any protein showing an undesirable degree of opalescence and/or forming high
molecular weight species can be used in accordance with the present invention. For
example, the present invention may be employed to reduce the opalescence of any pharmaceutrcally or commercially relevant antibody, receptor, cytokine, growth factor,
enzyme, clotting factor, hormone, regulatory factor, antigen, binding agent, among others,
The following list of proteins that can be separated according to the present invention is
merely exemplary in nature, and is not intended to be a limiting recitation. One of
ordinary skill in the art will understand that any protein may be expressed in accordance
with the present invention and will be able to select the particular protein to be produced
based as needed.
Antibodies and Binding Fragments
Antibodies, also known as immunoglobulins, are typically tetrameric glycosylated
proteins composed of two light (L) chains of approximately 25 kDa each and two heavy
(H) chains of approximately 50 kDa each. Two types of light chain, termed lambda and
kappa, may be found in antibodies. Depending on the amino acid sequence of the
constant domain of heavy chains, immunoglobulins can be assigned to five major classes:
A, D, E, G, and M, and several of these may be further divided into subclasses (isotypes),
e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Each light chain includes an N-terminal
variable (V) domain (VL) and a constant (C) domain (CL). Each heavy chain includes an
N-terminal V domain (VH), three or four C domains (CHs), and a hinge region. The CH
domain most proximal to VH is designated as CH1. The VH and VL domains consist of
four regions of relatively conserved sequences called framework regions (FR1, FR2, FR3,
and FR4), which form a scaffold for three regions of hypervariable sequences
(complementarity determining regions, CDRs). The CDRs contain most of the residues
responsible for specific interactions of the antibody with the antigen. CDRs are referred
to as CDR1, CDR2, and CDR3. Accordingly, CDR constituents on the heavy chain are
referred to as HI, H2, and H3, while CDR constituents on the light chain are referred to
as LI, L2, and L3. CDR3 is typically the greatest source of molecular diversity within
the antibody-binding site. H3, for example, can be as short as two amino acid residues or
greater than 26 amino acids. The subunit structures and three-dimensional configurations
of different classes of immunoglobulins are well known in the art. For a review of the

antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, eds. Harlow et al., 1988. One of skill in the art will recognize that each
subunit structure, e.g., a CH, VH, CL, VL, CDR, FR structure, comprises active
fragments, e.g., the portion of the VH, VL, or CDR subunit the binds to the antigen, i.e.,
the antigen-binding fragment, or, e.g., the portion of the CH subunit that binds to and/or
activates, e.g., an Fc receptor and/or complement. The CDRs typically refer to the Kabat
CDRs, as described in Sequences of Proteins of Immunological Interest, US Department
of Health and Human Services (1991), eds. Kabat et al. Another standard for
characterizing the antigen binding site is to refer to the hypervariable loops as described
by Chothia. See, e.g., Chothia, D. et al. (1992) J. Mol. Biol 227:799-817; and Tomlinson
et al. (1995) EMBO J. 14:4628-4638. Still another standard is the AbM definition used
by Oxford Molecular's AbM antibody modelling software. See, generally, e.g., Protein
Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody
Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag,
Heidelberg). Embodiments described with respect to Kabat CDRs can alternatively be
implemented using similar described relationships with respect to Chothia hypervariable
loops or to the AbM-defined loops.
Examples of binding fragments encompassed within the term "antigen-binding
fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of
the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd
fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL
and VH domains of a single arm of an antibody, (v) a dAb fragment, which consists of a
VH domain, (Vi) a camelld or camellzed variable domain, e.g., a VHH domain; (vii) a
single chain Fv (scFv); and (viii) a bispecific antibody. Furthermore, although the two
domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that enables them to be made as
a single protein chain in which the VL and VH regions pair to form monovalent
molecules (known as single chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-
26; Huston et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:5879-83). Such single chain
antibodies are also intended to be encompassed within the term "antigen-binding
fragment" of an antibody. These antibody fragments are obtained using conventional
techniques known to those skilled in the art, and the fragments are evaluated for function
in the same manner as are intact antibodies.

Other than "bispecific" or "bifunctional" antibodies, an antibody is understood to
have each of its binding sites identical. A "bispecific" or "bifunctional antibody" is an
artificial hybrid antibody having two different heavy/light chain pairs and two different
binding sites. Bispecific antibodies can be produced by a variety of methods including
fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann,
Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148,1547-1553
(1992).
Numerous methods known to those skilled in the art are available for obtaining
antibodies. For example, monoclonal antibodies may be produced by generation of
hybridomas in accordance with known methods. Hybridomas formed in this manner are
then screened using standard methods, such as enzyme-linked immunosorbent assay
(ELISA) and surface plasmon resonance (BIACORE™) analysis, to identify one or more
hybridomas that produce an antibody that specifically binds with a specified antigen. Any
form of the specified antigen may be used as the immunogen, e.g., recombinant antigen,
naturally occurring forms, any variants or fragments thereof, as well as antigenic peptide
thereof.
One exemplary method of making antibodies includes screening protein
expression libraries, e.g., phage or ribosome display libraries. Phage display is described,
for example, in Ladner et al., U.S. Patent No. 5,223,409; Smith (1985) Science 228:1315-
1317; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO
92/01047; WO 92/09690; and WO 90/02809.
In addition to the use of display libraries, the specified antigen can be used to
immunize a non-human animal, e.g., a rodent, e.g., a mouse, hamster, or rat. In one
ombodiment, the non-numan animal includes at least a part of a human immunoglobulin
gene. For example, it is possible to engineer mouse strains deficient in mouse antibody
production with large fragments of the human Ig loci. Using the hybridoma technology,
antigen-specific monoclonal antibodies derived from the genes with the desired
specificity may be produced and selected. See, e.g., XENOMOUSE™, Green et al.
(1994) Nature Genetics 7:13-21, US 2003-0070185, WO 96/34096, published Oct. 31,
1996, and PCT Application No. PCT/US96/05928, filed Apr. 29, 1996.
In another embodiment, a monoclonal antibody is obtained from the non-human
animal, and then modified, e.g., humanized, deimmunized, chimeric, may be produced
using recombinant DNA techniques known in the art. A variety of approaches for making
chimeric antibodies have been described. See e.g., Morrison et al, Proc. Natl. Acad. Sci.

U.S.A. 81:6851, 1985; Takeda et ai, Nature 314:452, 1985, Cabilly et ai, U.S. Patent No.
4,816,567; Boss et ai, U.S. Patent No. 4,816,397; Tanaguchi et a!., European Patent
Publication EP171496; European Patent Publication 0173494, United Kingdom Patent
GB 2177096B. Humanized antibodies may also be produced, for example, using
transgenic mice that express human heavy and light chain genes, but are incapable of
expressing the endogenous mouse immunoglobulin heavy and light chain genes. Winter
describes an exemplary CDR-grafting method that may be used to prepare the humanized
antibodies described herein (U.S. Patent No. 5,225,539). All of the CDRs of a particular
human antibody may be replaced with at least a portion of a non-human CDR, or only
some of the CDRs may be replaced with non-human CDRs. It is only necessary to
replace the number of CDRs required for binding of the humanized antibody to a
predetermined antigen.
Humanized antibodies can be generated by replacing sequences of the Fv variable
domain that are not directly involved in antigen binding with equivalent sequences from
human Fv variable domains. Exemplary methods for generating humanized antibodies or
fragments thereof are provided by Morrison (1985) Science 229:1202-1207; by Oi et al.
(1986) BioTechniques 4:214; and by US 5,585,089; US 5,693,761; US 5,693,762; US
5,859,205; and US 6,407,213. Those methods include isolating, manipulating, and
expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv
variable domains from at least one of a heavy or light chain. Such nucleic acids may be
obtained from a hybridoma producing an antibody against a predetermined target, as
described above, as well as from other sources. The recombinant DNA encoding the
humanized antibody molecule can then be cloned into an appropriate expression vector.
In certain embodiments, a humanized antibody is optimized by the introduction of
conservative substitutions, consensus sequence substitutions, germline substitutions
and/or backmutations. Such altered immunoglobulin molecules can be made by any of
several techniques known in the art, (e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80:
7308-7312, 1983;Kozbor et al, Immunology Today, 4: 7279, 1983; Olsson etal.,Meth.
Enzymol., 92: 3-16, 1982), and may be made according to the teachings of PCT
Publication WO92/06193 or EP 0239400).
An antibody may also be modified by specific deletion of human T cell epitopes
or "deimmunization" by the methods disclosed in WO 98/52976 and WO 00/34317.
Briefly, the heavy and light chain variable domains of an antibody can be analyzed for
peptides that bind to MHC Class II; these peptides represent potential T-cell epitopes (as

defined in WO 98/52976 and WO 00/34317). For detection of potential T-cell epitopes, a
computer modeling approach termed "peptide threading" can be applied, and in addition a
database of human MHC class II binding peptides can be searched for motifs present in
the VH and VL sequences, as described in WO 98/52976 and WO 00/34317. These motifs
bind to any of the 18 major MHC class IIDR allotypes, and thus constitute potential T
cell epitopes. Potential T-cell epitopes detected can be eliminated by substituting small
numbers of amino acid residues in the variable domains, or preferably, by single amino
acid substitutions. Typically, conservative substitutions are made. Often, but not
exclusively, an amino acid common to a position in human germline antibody sequences
may be used. Human germline sequences, e.g., are disclosed in Tbmlinson, et al. (1992)
J. Mol. Biol. 227:776-798; Cook, G P. et al. (1995) Immunol. Today Vol. 16 (5): 237-242;
Chothia, D. et al. (1992) J. Mol. Biol. 227:799-817; and Tomlinson et al. (1995) EMBOJ.
14:4628-4638. The V BASE directory provides a comprehensive directory of human
immunoglobulin variable region sequences (compiled by Tomlinson, I.A. et al. MRC
Centre for Protein Engineering, Cambridge, UK). These sequences can be used as a
source of human sequence, e.g., for framework regions and CDRs. Consensus human
framework regions can also be used, e.g., as described in U.S. 6,300,064.
In certain embodiments, an antibody can contain an altered immunoglobulin
constant or Fc region. For example, an antibody produced in accordance with the
teachings herein may bind more strongly or with more specificity to effector molecules
such as complement and/or Fc receptors, which can control several immune functions of
the antibody such as effector cell activity, lysis, complement-mediated activity, antibody
clearance, and antibody half-life. Typical Fc receptors that bind to an Fc region of an
antibody (e.g., an igG antibody) include, but are not limited to, receptors of the FcγRI,
FcγRII, and FcγRIII and FcRn subclasses, including allelic variants and alternatively
spliced forms of these receptors. Fc receptors are reviewed in Ravetch and Kinet, Annu.
Rev. Immunol 9:457-92, 1991; Capel et al., Immunomethods 4:25-34,1994; and de Haas
et al.,J. Lab. Clin. Med. 126:330-41, 1995).
Non-limiting examples of antibodies that can be separated by the methods of the
invention, include but are not limited to, antibodies against Ap, IL-13, IL-22, GDF8 and
5T4. Each of these antibodies is described in more detail hereinbelow and the appended
Examples.

Anti-GDF8 Antibodies
Exemplary antibodies that can be used in the methods of the invention are anti-
GDF8 antibodies. The term "GDF-8" refers to growth and differentiation factor-8 and,
where appropriate, factors that are structurally or functionally related to GDF-8, for
example, BMP-11 and other factors belonging to the TGF-β superfamily. The term refers
to the full-length unprocessed precursor form of GDF-8, as well as the mature and
propeptide forms resulting from post-translational cleavage. The term also refers to any
fragments and variants of GDF-8 that maintain at least some biological activities
associated with mature GDF-8, including sequences that have been modified. The amino
acid sequence human GDF- 8, as well as many other vertebrate species (including
murine, baboon, bovine, chicken) is disclosed, e.g., U.S. 04/0142382, US 02/0157125,
and McPherron et al. (1997) Proc. Nat. Acad. Sci. U.S.A., 94:12457-12461). Examples
of neutralizing antibodies against GDF-8, e.g., Myo-029, are disclosed in, e.g., U.S.
2004/0142382, and are referenced throughout the Examples appended herein. Exemplary
disease and disorders include muscle and neuromuscular disorders such as muscular
dystrophy (including Duchenne's muscular dystrophy); amyotrophic lateral sclerosis;
muscle atrophy; organ atrophy; frailty; tunnel syndrome; congestive obstructive
pulmonary disease; sarcopenia, cachexia, and other muscle wasting syndromes; adipose
tissue disorders (e.g., obesity); type 2 diabetes; impaired glucose tolerance; metabolic
syndromes (e.g., syndrome X); insulin resistance induced by trauma such as bums or
nitrogen imbalance; and bone degenerative diseases {e.g., osteoarthritis and osteoporosis).
Anti-Aβ Antibodies
A reduction in the opalescence of anti-Ap antibody preparations can be practiced
following the teachings of the invention. The terms "AB antibody," "Ap antibody,"
"anti-AP antibody," and "anti-AP" are used interchangeably herein to refer to an antibody
that binds to one or more epitopes or antigenic determinants of APP, Ap protein, or both.
Exemplary epitopes or antigenic determinants can be found within the human amyloid
precursor protein (APP), but are preferably found within the Ap peptide of APP.
Multiple isoforms of APP exist, for example APP695, APP751, and APP770. Amino acids
within APP are assigned numbers according to the sequence of the APP770 isoform (see
e.g., GenBank Accession No. P05067). Ap (also referred to herein as beta amyloid
peptide and A beta) peptide is a ~4-kDa internal fragment of 39-43 amino acids of APP

(Aβ39, Aβ40, Aβ41, Aβ42, and Aβ43). Aβ40, for example, consists of residues 672-711
of APP and Aβ42 consists of residues 672-713 of APP. As a result of proteolytic
processing of APP by different secretase enzymes iv vivo or in situ, Aβ is found in both a
"short form," 40 amino acids in length, and a "long form," ranging from 42-43 amino
acids in length. Epitopes or antigenic determinants can be located within the N-terminus
of the Aβ peptide and include residues within amino acids 1-10 of Ap, preferably from
residues 1-3, 1-4, 1-5, 1-6, 1-7, 2-7, 3-6, or 3-7 of Aβ42 or within residues 2-4, 5, 6, 7, or
8 of Ap, residues 3-5, 6, 7, 8, or 9 of Ap, or residues 4-7, 8, 9, or 10 of Aβ42. "Central"
epitopes or antigenic determinants are located within the central or mid-portion of the Aβ
peptide and include residues within amino acids 16-24, 16-23, 16-22,16-21, 19-21, 19-
22, 19-23, or 19-24 of Aβ. "C-terminal" epitopes or antigenic determinants are located
within the C-terminus of the Aβ peptide and include residues within amino acids 33-40,
33-41, or 33-42 of Aβ.
In various embodiments, an Ap antibody is end-specific. As used herein, the term
"end-specific" refers to an antibody which specifically binds to the N-terminal or C-
terminal residues of an Aβ peptide but that does not recognize the same residues when
present in a longer Aβ species comprising the residues or in APP.
In various embodiments, an Aβ antibody is "C-terminus-specific." As used
herein, the term "C terminus-specific" means that the antibody specifically recognizes a
free C-terminus of an Aβ peptide. Examples of C terminus-specific Aβ antibodies
include those that: recognize an Aβ peptide ending at residue 40, but do not recognize an
Ap peptide ending at residue 41,42, and/or 43; recognize an Aβ peptide ending at residue
42, but ao not recognize an Aβ peptide ending at residue 40, 41, and/or 43; etc.
In one embodiment, the antibody may be a 3D6 antibody or variant thereof, or a
10D5 antibody or variant thereof, both of which are described in U.S. Patent Publication
No. 2003/0165496A1, U.S. Patent Publication No. 2004/0087777A1, International Patent
Publication No. WO02/46237A3. Description of 3D6 and 10D5 can also be found, for
example, in International Patent Publication No. WO02/088306A2 and International
Patent Publication No. WO02/088307A2. 3D6 is a monoclonal antibody (mAb) that
specifically binds to an N-terminal epitope located in the human β-amyloid peptide,
specifically, residues 1-5. By comparison, 10D5 is a mAb that specifically binds to an N-
terminal epitope located in the human β-amyloid peptide, specifically, residues 3-6. In
another embodiment, the antibody may be a 12B4 antibody or variant thereof, as

described in U.S. Patent Publication No. 20040082762A1 and International Patent
Publication No. WO03/077858A2. 12B4 is a mAb that specifically binds to an N-
terminal epitope located in the human β-amyloid peptide, specifically, residues 3-7. In
yet another embodiment, the antibody may be a 12A11 antibody or a variant thereof, as
described in U.S. Patent Application No. 10/858,855 and International Patent Application No. PCT/US04/17514. 12A11 is a mAb that specifically binds to an N-terminal epitope
located in the human β-amyloid peptide, specifically, residues 3-7. In yet another
embodiment, the antibody may be a 266 antibody as described in U.S. Patent Application
No. 10/789,273, and International Patent Application No. WO01/62801A2. Antibodies
designed to specifically bind to C-terminal epitopes located in human β-amyloid peptide,
for use in the present invention include, but are not limited to, 369.2B, as described in
U.S. Patent No. 5,786,160.
In exemplary embodiments, the antibody is a humanized anti Aβ peptide 3D6
antibody that selectively binds Aβ peptide. More specifically, the humanized anti Aβ
peptide 3D6 antibody is designed to specifically bind to an NH2-terminal epitope located
in the human β-amyloid 1-40 or 1-42 peptide found in plaque deposits in the brain (e.g.,
in patients suffering from Alzheimer's disease).
Anti-Aβ antibodies can be used to treat amyloidogenic diseases, in particular,
Alzheimer's Disease. The term "amyloidogenic disease" includes any disease associated
with (or caused by) the formation or deposition of insoluble amyloid fibrils. Exemplary
amyloidogenic diseases include, but are not limited to, systemic amyloidosis, Alzheimer's
disease, mature onset diabetes, Parkinson's disease, Huntington's disease, fronto-
temporal dementia, and the prion-related transmissible spongiform encephalopathies
(kuru and Creuizfeldt-Jacob disease in humans and scrapie and BSE in sheep and cattle,
respectively). Different amyloidogenic diseases are defined or characterized by the
nature of the polypeptide component of the fibrils deposited. For example, in subjects or
patients having Alzheimer's disease, P-amyloid protein (e.g., wild-type, variant, or
truncated p-amyloid protein) is the characterizing polypeptide component of the amyloid
deposit. Accordingly, Alzheimer's disease is an example of a "disease characterized by
deposits of Aβ" or a "disease associated with deposits of Aβ," e.g., in the brain of a
subject or patient. The terms "p-amyloid protein," "P-amyloid peptide," "p-amyloid,"
"Aβ," and "Aβ peptide" are used interchangeably herein.

Anti-5T4 Antibodies
The 5T4 antigen has been previously characterized (see e.g., WO 89/07947). The
full nucleic acid sequence of human 5T4 is known (Myers et al. (1994) J Biol Chem 169:
9319-24 and GenBank at Accession No. Z29083). The sequence for 5T4 antigen from
other species is also known, for example, murine 5T4 (WOOO/29428), canine 5T4
(WO01/36486) or feline 5T4 ( US 05/0100958).
Human 5T4 is a glycoprotein of about 72 kDa expressed widely in carcinomas,
but having a highly restricted expression pattern in normal adult tissues. It appears to be
strongly correlated to metastasis in colorectal and gastric cancer. Expression of the 5T4
antigen is also found at high frequency in breast and ovarian cancers (Starzynska et al.
(1998) Eur. J. Gastroenterol. Hepatol. 10:479-84; Starzynska et al. (1994) Br. J. Cancer
69:899-902; Starzynska et al. (1992) Br. J. Cancer 66:867-9). 5T4 has been proposed as
a marker, with possible mechanistic involvement, for tumor progression and metastasis
potential (Carsberg et al. (1996) Int J Cancer 68:84-92). 5T4 has also been proposed for
use as an immunotherapeutic agent (see WO 00/29428). Antigenic peptides of 5T4 are
disclosed in, e.g., US 05/0100958, the contents of which are incorporated by reference.
Several pending applications relate generally to nucleic acids encoding the anti-
5T4 monoclonal antibody, vectors and host cells thereof, for example, U.S. Application
Publication Nos. 2003/0018004 and 2005/0032216. A provisional patent application
pertaining generally to the humanized anti-5T4 H8 monoclonal antibodies and
calicheamicin conjugates thereof, as well as methods of treatment using these
calicheamicin conjugates has been filed (U.S. Application Publication No.
2006/0088522). The contents of all of these applications are incorporated by reference
herein in their enthety.
Anti-IL13 Antibodies
Interleukin-13 (IL-13) is a previously characterized cytokine secreted by
T lymphocytes and mast cells (McKenzie et al. (1993) Proc. Natl. Acad. Sci. USA
90:3735-39; Bost et al. (1996) Immunology 87:663-41). The term "IL-13" refers to
interleukin-13, including full-length unprocessed precursor form of IL-13, as well as the
mature forms resulting from post-translational cleavage. The term also refers to any
fragments and variants of IL-13 that maintain at least some biological activities associated
with mature IL-13, including sequences that have been modified. The term 'TL-13"
includes human IL-13, as well as other vertebrate species. Several pending applications

disclose antibodies against human and monkey IL-13, IL-13 peptides, vectors and host
cells producing the same, for example, U.S. Application Publication Nos.
2006/0063228A and 2006/0073148. The contents of all of these publications are
incorporated by reference herein in their entirety.
IL-13 shares several biological activities with IL-4. For example, either IL-4 or
IL-13 can cause IgE isotype switching in B cells (Tomkinson et al. (2001) J. Immunol.
166:5792-5800). Additionally, increased levels of cell surface CD23 and serum CD23
(sCD23) have been reported in asthmatic patients (Sanchez-Guererro et al. (1994) Allergy
49:587-92; DiLorenzo et al. (1999) Allergy Asthma Proc. 20:119-25). In addition, either
IL-4 or IL-13 can upregulate the expression of MHC class II and the low-affinity IgE
receptor (CD23) on B cells and monocytes, which results in enhanced antigen
presentation and regulated macrophage function (Tomkinson et al., supra). These
observations suggest that IL-13 may be an important player in the development of airway
eosinophilia and airway hyperresponsiveness (AHR) (Tomkinson et al., supra; Wills-
Karp et al. (1998) Science 282:2258-61). Accordingly, inhibition of IL-13 can be useful
in ameliorating the pathology of a number of inflammatory and/or allergic conditions,
including, but not limited to, respiratory disorders, e.g., asthma; chronic obstructive
pulmonary disease (COPD); other conditions involving airway inflammation,
eosinophilia, fibrosis and excess mucus production, e.g., cystic fibrosis and pulmonary
fibrosis; atopic disorders, e.g., atopic dermatitis, urticaria, eczema, allergic rhinitis;
inflammatory and/or autoimmune conditions of, the skin (e.g., atopic dermatitis),
gastrointestinal organs (e.g., inflammatory bowel diseases (IBD), such as ulcerative
colitis and/or Crohn's disease), liver (e.g., cirrhosis, hepatocellular carcinoma);
scierodenna; tumors or cancers (e.g., soft tissue or solid tumors), such as leukemia,
glioblastoma, and lymphoma, e.g., Hodgkin's lymphoma; viral infections (e.g., from
HTLV-1); fibrosis of other organs, e.g., fibrosis of the liver, (e.g., fibrosis caused by a
hepatitis B and/or C virus).
Anti-IL22 Antibodies
Interleukin-22 (IL-22) is a previously characterized class II cytokine that shows
sequence homology to IL-10. Its expression is up-regulated in T cells by IL-9 or ConA
(Dumoutier L. et al. (2000) Proc Natl Acad Sci USA 97(18):10144- 9). Studies have
shown that expression of IL-22 mRNA is induced in vivo in response to LPS
administration, and that IL-22 modulates parameters indicative of an acute phase response

(Dumoutier L. et al. (2000) supra; Pittman D. et al. (2001) Genes and Immunity 2:172),
and that a reduction of IL-22 activity by using a neutralizing anti-IL-22 antibody
ameliorates inflammatory symptoms in a mouse collagen-induced arthritis (CIA) model.
Thus, IL-22 antagonists, e.g., neutralizing anti-IL-22 antibodies and fragments thereof,
can be used to induce immune suppression in vivo, for examples, for treating autoimmune
disorders (e.g., arthritic disorders such as rheumatold artnntis); respiratory disorders (e.g.,
asthma, chronic obstructive pulmonary disease (COPD)); inflammatory conditions of,
e.g., the skin (e.g., psoriasis), cardiovascular system (e.g., atherosclerosis), nervous
system (e.g., Alzheimer's disease), kidneys (e. g., nephritis), liver (e.g., hepatitis) and
pancreas (e.g., pancreatitis).
The term "IL-22" refers to interleukin-22, including full-length unprocessed
precursor form of IL-22, as well as the mature forms resulting from post-translational
cleavage. The term also refers to any fragments and variants of IL-22 that maintain at
least some biological activities associated with mature IL-22, including sequences that
have been modified. The term "IL-22" includes human IL-22, as well as other vertebrate
species. The amino acid and nucleotide sequences of human and rodent IL-22, as well as
antibodies against IL-22 are disclosed in, for example, U.S. Application Publication Nos.
2005-0042220 and 2005-0158760, and U.S. Patent No. 6,939,545. The contents of all of
these publications are incorporated by reference herein in their entirety.
Small Modular [mmunoPharmaceuticals (SMIP )
The present invention can also be applied to Small Modular
ImmunoPharmaceuticals (SMIP™). It typically refers to a binding domain-fusion protein
that includes a binding domain polypeptide that is rused or otnerwise connected to an
immunoglobulin hinge or hinge-acting region polypeptide, which in turn is fused or
otherwise connected to a region comprising one or more native or engineered constant
regions from an immunoglobulin heavy chain, other than CH1, for example, the CH2 and
CH3 regions of IgG and IgA, or the CH3 and CH4 regions of IgE (see e.g., U.S.
05/0136049 by Ledbetter, J. et al. for a more complete description). The binding domain-
immunoglobulin fusion protein can further include a region that includes a native or
engineered immunoglobulin heavy chain CH2 constant region polypeptide (or CH3 in the
case of a construct derived in whole or in part from IgE) that is fused or otherwise
connected to the hinge region polypeptide and a native or engineered immunoglobulin
heavy chain CH3 constant region polypeptide (or CH4 in the case of a construct derived

in whole or in part from IgE) that is fused or otherwise connected to the CH2 constant
region polypeptide (or CH3 in the case of a construct derived in whole or in part from
IgE). Typically, such binding domain-irnmunoglobulin fusion proteins are capable of at
least one immunological activity selected from the group consisting of antibody
dependent cell-mediated cytotoxicity, complement fixation, and/or binding to a target, for
example, a target antigen.
Soluble Receptors and Receptor Fusions
The invention can also be applied to soluble receptors or fragments thereof.
Examples of soluble receptors include the extracellular domain of a receptor, such as
soluble tumor necrosis factor alpha and beta receptors (TNFR-1; EP 417,563 published
Mar. 20, 1991; TNFR-2, EP 417,014 published Mar. 20, 1991; and reviewed in Naismith
and Sprang, J Inflamm. 47(l-2):l-7, 1995-96, each of which is incorporated herein by
reference in its entirety). In other embodiments, the soluble receptor includes the
extracellular domain of interleukin-21 receptor (IL-21R) as described in, for example, US
2003-0108549 (the contents of which are also incorporated by reference).
The fusion protein can include a targeting moiety, e.g., a soluble receptor
fragment or a ligand, and an immunoglobulin chain, an Fc fragment, a heavy chain
constant regions of the various isotypes, including: IgGl, IgG2, IgG3, IgG4, IgM, IgAl,
IgA2, IgD, and IgE. For example, the fusion protein can include the extracellular domain
of a receptor, and, e.g., fused to, a human immunoglobulin Fc chain (e.g., human IgG,
e.g., human IgGl or human IgG4, or a mutated form thereof). In one embodiment, the
human Fc sequence has been mutated at one or more amino acids, e.g., mutated at
residues 254 and 257 from the wild type sequence to reduce Fc receptor binding. The
fusion proteins may additionally include a linker sequence joining the first moiety to the
second moiety, e.g., the immunoglobulin fragment. For example, the fusion protein can
include a peptide linker, e.g., a peptide linker of about 4 to 20, more preferably, 5 to 10,
amino acids in length; the peptide linker is 8 amino acids in length. For example, the
fusion protein can include a peptide linker having the formula (Ser-GIy-Gly-Gly-Gly)y
wherein y is 1, 2, 3, 4, 5, 6, 7, or 8. In other embodiments, additional amino acid
sequences can be added to the N- or C-terminus of the fusion protein to facilitate
expression, steric flexibility, detection and/or isolation or purification.
In certain embodiments, the soluble receptor fusion comprises a soluble TNFR-Ig
(e.g., a soluble fragment of a TNF receptor, e.g., p55 or p75 human TNF receptor or

derivatives thereof, e.g., 75 kd TNFR-IgG (e.g., 75 kD TNF receptor fused to a 235
amino acid Fc portion of human IgGl).
A chimeric or fusion protein of the invention can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for the different
polypeptide sequences are ligated together in-frame in accordance with conventional
techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation,
restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends
as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and
enzymatic ligation. In another embodiment, the fusion gene can be synthesized by
conventional techniques including automated DNA synthesizers. Alternatively, PCR
amplification of gene fragments can be carried out using anchor primers that give rise to
complementary overhangs between two consecutive gene fragments that can
subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for
example, Ausubel et al. (eds.) Current Protocols in Molecular Biology, John Wiley &
Sons, 1992). Moreover, many expression vectors are commercially available that encode
a fusion moiety (e.g., an Fc region of an immunoglobulin heavy chain). Immunoglobulin
fusion polypeptides are known in the art and are described in e.g., U.S. Pat. Nos.
5,516,964; 5,225,538; 5,428,130; 5,514,582; 5,714,147; and 5,455,165.
Growth Factors and Cytokines
Another class of polypeptides that have been shown to be effective as
pharmaceutical and/or commercial agents and that can desirably be produced according to
the teachings of the present invention includes growth factors and other signaling
includes, such as cytokines.
Growth factors are typically glycoproteins that are secreted by cells and bind to
and activate receptors on other cells, initiating a metabolic or developmental change in
the receptor cell. Non-limiting examples of mammalian growth factors and other
signaling molecules include cytokines; epidermal growth factor (EGF); platelet-derived
growth factor (PDGF); fibroblast growth factors (FGFs) such as aFGF and bFGF;
transforming growth factors (TGFs) such as TGF-alpha and TGF-beta, including TGF-
beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, or TGF-beta 5; insulin-like growth factor-I
and -II (IGF-I and IGF-II); des(l-3) -IGF-I (brain IGF-I), insulin-like growth factor
binding proteins; CD proteins such as CD-3, CD-4, CD-8, and CD-19; erythropoietin;
osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an

interferon such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs),
e.g., M-CSF, GM-CSF, and G-CSF; interleukins (TLs), e.g., IL-1 to IL-13 (e.g., IL-11);
tumor necrosis factor (TNF) alpha and beta; insulin A-chain; insulin B-chain; proinsulin;
follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors
such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anti-clotting
factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen
activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA);
bombesin; thrombin, hemopoietic growth factor; enkephalinase; RANTES (regulated on
activation normally T-cell expressed and secreted); human macrophage inflammatory
protein (MIP-1-alpha); mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain;
prorelaxin; mouse gonadotropin-associated peptide; neurotrophic factors such as bone-
derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or
NT-6), or a nerve growth factor such as NGF-beta. One of ordinary skill in the art will be
aware of other growth factors or signaling molecules that can be expressed in accordance
with methods and compositions of the present invention.
Specific alterations in the glycosylation pattern of growth factors or other
signaling molecules have been shown to have dramatic effects on their therapeutic
properties. As one example, a common method of treatment for patients who suffer from
chronic anemia is to provide them with frequent injections of recombinant human
erythropopietin (rHuEPO) in order to boost their production of red blood cells. An
analog of rHuEPO, darbepoetin alfa (ARANESP®), has been developed to have a longer
duration than normal rHuEPO. The primary difference between darbepoetin alfa and
rHuEPO is the presence of two extra sialic-acid-containing N-linked oligosaccharide
chains, .production of darbepoetin alfa has been accomplished using in vitro
glycoengineering (see Elliott et al., Nature Biotechnology 21(4):414-21, 2003,
incorporated herein by reference in its entirety). Elliott et al. used in vitro mutagenesis to
incorporate extra glycosylation sites into the rHuEPO polypeptide backbone, resulting in
expression of the darbepoetin alfa analog. The extra oligosaccharide chains are located
distal to the EPO receptor binding site and apparently do not interfere with receptor
binding. However, darbepoetin alfa's half-life is up to three-fold higher than rHuEPO,
resulting in a much more effective therapeutic agent.

Clotting Factors
Clotting factors have been shown to be effective as pharmaceutical and/or
commercial agents. Hemophilia B is a disorder in which the blood of the sufferer is
unable to clot. Thus, any small wound that results in bleeding is potentially a life-
threatening event. For example, Coagulation Factor IX (Factor IX or "FIX") is a single-
chain glycoprotein whose deficiency results in Hemophilia B. FIX is synthesized as a
single chain zymogen that can be activated to a two-chain serine protease (Factor IXa) by
release of an activation peptide. The catalytic domain of Factor IXa is located in the
heavy chain (see Chang et al., J. Clin. Invest., 100:4, 1997, incorporated herein by
reference in its entirety). FIX has multiple glycosylation sites including both N-linked
and O-linked carbohydrates. One particular O-linked structure at Serine 61 (Sia-α2,3-
Gal-β1,4-GlcNAc-β1,3-Fuc-αl-0-Ser) was once thought unique to FIX but has since
found on a few other molecules including the Notch protein in mammals and Drosophila
(Maloney et al, Journal of Biol. Chem., 275(13), 2000). FIX produced by Chinese
Hamster Ovary ("CHO") cells in cell culture exhibits some variability in the Serine 61
oligosaccharide chain. These different glycoforms, and other potential glycoforms, may
have different abilities to induce clotting when administered to humans or animals and/or
may have different stabilities in the blood, resulting in less effective clotting.
Hemophilia A, which is clinically indistinguishable from Hemophilia B, is caused
by a defect in human clotting factor VIII, another glycoprotein that is synthesized as a
single chain and then processed into a two-chain active form. The present invention may
also be employed to control or alter the glycosylation pattern of clotting factor VIII in
order to modulate its clotting activity. Other clotting factors that can be produced in
accordance with the present invention include tissue factor and von Willebrands factor.
Enzymes
Another class of polypeptides that have been shown to be effective as
pharmaceutical and/or commercial agents and that can desirably be produced according to
the teachings of the present invention includes enzymes. Enzymes may be glycoproteins
whose glycosylation pattern affects enzymatic activity. Thus, the present invention may
also be used to produce enzymes in a cell culture wherein the produced enzymes have a
more extensive or otherwise more desirable glycosylation pattern.
As but one non-limiting example, a deficiency in glucocerebrosidase (GCR)
results in a condition known as Gaucher's disease, which is caused by an accumulation of

glucocerebrosidase in lysosomes of certain cells. Subjects with Gaucher's disease exhibit
a range of symptoms including splenomegaly, hepatomegaly, skeletal disorder,
thrombocytopenia and anemia. Friedman and Hayes showed that recombinant GCR
(rGCR) containing a single substitution in the primary amino acid sequence exhibited an
altered glycosylation pattern, specifically an increase in fucose and N-acetyl glucosamine
residues" compared "to naturally occurring GCR (see United States Patent number
5,549,892).
Friedman and Hayes also demonstrated that this rGCR exhibited improved
pharmacokinetic properties compared to naturally occurring rGCR. For example,
approximately twice as much rGCR targeted liver Kupffer cells than did naturally
occurring GCR. Although the primary amino acid sequences of the two proteins differed
at a single residue, Friedman and Hayes hypothesized that the altered glycosylation
pattern of rGCR may also influence the targeting to Kupffer cells. One of ordinary skill
in the art will be aware of other known examples of enzymes that exhibit altered
enzymatic, pharmacokinetic and/or pharmacodynamic properties resulting from an
alteration in their glycosylation patterns.
Protein Production
Recombinant methods of producing the proteins according to the invention are
known in the art. Nucleotide sequences encoding the proteins are typically inserted in an
expression vector for introduction into host cells that may be used to produce the desired
quantity of modified antibody that, in turn, provides the polypeptides. The term "vector"
includes a nucleic acid construct often including a nucleic acid, e.g., a gene, and further
including mininal elements necessary for nucleic acia replication, transcription, stability
and/or protein expression or secretion from a host cell. Such constructs may exist as
extrachromosomal elements or may be integrated into the genome of a host cell.
The term "expression vector" includes a specific type of vector wherein the
nucleic acid construct is optimized for the high-level expression of a desired protein
product. Expression vectors often have transcriptional regulatory agents, such as
promoter and enhancer elements, optimized for high-levels of transcription in specific cell
types and/ or optimized such that expression is constitutive based upon the use of a
specific inducing agent. Expression vectors further have sequences that provide for
proper and/or enhanced translation of the protein As known to those skilled in the art,
such vectors may easily be selected from the group consisting of plasmids, phages,

viruses, and retroviruses. The term "expression cassette" includes a nucleic acid
construct containing a gene and having elements in addition to the gene that allow for
proper and or enhanced expression of that gene in a host cell. For producing antibodies,
nucleic acids encoding light and heavy chains can be inserted into expression vectors.
Such sequences can be present in the same nucleic acid molecule (e.g., the same
expression vector) or alternatively, can be expressed from separate nucleic acid molecules
(e.g., separate expression vectors).
The term "operably linked" includes a juxtaposition wherein the components are
in a relationship permitting them to function in their intended manner (e.g., functionally
linked). As an example, a promoter/enhancer operably linked to a polynucleotide of
interest is ligated to said polynucleotide such that expression of the polynucleotide of
interest is achieved under conditions which activate expression directed by the
promoter/enhancer.
Expression vectors are typically replicable in the host organisms either as
episomes or as an integral part of the host chromosomal DNA. Commonly, expression
vectors contain selection markers (e.g., ampicillin-resistance, hygrbmycin-resistance,
tetracycline resistance, kanamycin resistance or neomycin resistance) to permit detection
of those cells transformed with the desired DNA sequences (see, e.g., Itakura et al., U.S.
Patent No. 4,704,362). In addition to the immunoglobulin DNA cassette sequences,
insert sequences, and regulatory sequences, the recombinant expression vectors of the
invention may carry additional sequences, such as sequences that regulate replication of
the vector in host cells (e.g., origins of replication) and selectable marker genes. The
selectable marker gene facilitates selection of host cells into which the vector has been
introduced (see e.g., US. Pat. nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et
al.). For example, typically the selectable marker gene confers resistance to drugs, such
as G418, hygromycin, or methotrexate, on a host cell into which the vector has been
introduced. Preferred selectable marker genes include the dihydrofolate reductase
(DHFR) gene (for use in dhfr host cells with methotrexate selection/amplification) and
the neo gene (for G418 selection).
Once the vector has been incorporated into the appropriate host cell, the host cell
is maintained under conditions suitable for high level expression of the nucleotide
sequences, and the collection and purification of the desired antibodies. Any host cell
susceptible to cell culture, and to expression of proteins or polypeptides, may be utilized
in accordance with the present invention. In certain embodiments, the host cell is

mammalian. Non-limiting examples of mammalian cells that may be used in accordance
with the present invention include BALB/c mouse myeloma line (NSO/1, ECACC No:
85110503); human retinoblasts (PER.C6, CruCell, Leiden, The Netherlands); monkey
kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic
kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J.
Gen Virol., 36:59,1977); baby hamster kidney cells(BHK, ATCC CCL 10); Chinese
hamster ovary cells +/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,
77:4216, 1980); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251, 1980);
monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-
76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine
kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL
1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065);
mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals
N.Y. Acad. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and a human hepatoma line
(Hep G2).
Additionally, any number of commercially and non-commercially available
hybridoma cell lines that express polypeptides or proteins may be utilized in accordance
with the present invention. One skilled in the art will appreciate that hybridoma cell lines
might have different nutrition requirements and/or might require different culture
conditions for optimal growth and polypeptide or protein expression, and will be able to
modify conditions as needed.
Expression vectors for these cells can include expression control sequences, such
as an origin of replication, a promoter, and an enhancer (Queen et al., Immunol. Rev.
80:10(1986)), and necessary processing information sites, such as ribosome binding sites,
RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
Preferred expression control sequences are promoters derived from immunoglobulin
genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus and the like. (See,
e.g., Co et al., (1992) J. Immunol. 148:1149). Preferred regulatory sequences for
mammalian host cell expression include viral elements that direct high levels of protein
expression in mammalian cells, such as promoters and/or enhancers derived from FF-1a
promoter and BGH poly A, cytomegalovirus (CMV) (such as the CMV promoter/
enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus
(e.g., the adenovirus major late promoter (AdMLP)), and polyoma. For further
description of viral regulatory elements, and sequences thereof, see, e.g., U.S. Pat. No.

5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al. and U.S. Patent No.
4,968,615 by Schaffner et al. In exemplary embodiments, the antibody heavy and light
chain genes are operatively linked to enhancer/promoter regulatory elements (e.g.,
derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLP
promoter regulatory element or an SV40 enhancer/AdMLP promoter regulatory element)
to drive high levels of transcription of the genes. In exemplary embodiments of the
invention, the construct include an internal ribosome entry site (IRES) to provide
relatively high levels of polypeptides of the invention in eukaryotic host cells.
Compatible IRES sequences are disclosed in U.S. Pat. No. 6,193,980 that is also
incorporated herein.
Alternatively, coding sequences can be incorporated in a transgene for
introduction into the genome of a transgenic animal and subsequent expression in the
milk of the transgenic animal {see, e.g., Deboer et al., US 5,741,957, Rosen, US
5,304,489, and Meade et al., US 5,849,992). Suitable transgenes include coding
sequences for light and/or heavy chains in operable linkage with a promoter and enhancer
from a mammary gland specific gene, such as casein or beta lactoglobulin.
Prokaryotic host cells may also be suitable for producing the antibodies of the
invention. E. coli is one prokaryotic host particularly useful for cloning the
polynucleotides {e.g., DNA sequences) of the present invention. Other microbial hosts
suitable for use include bacilli, such as Bacillus subtilis, enterobacteriaceae, such as
Escherichia, Salmonella, and Serratia, and various Pseudomonas species. In these
prokaryotic hosts, one can also make expression vectors, which will typically contain
expression control sequences compatible with the host cell {e.g., an origin of replication).
In addition, any number of a variety oi well-known promoters will be present, such as the
lactose promoter system, a tryptophan (tip) promoter system, a beta-lactamase promoter
system, or a promoter system from phage lambda. The promoters will typically control
expression, optionally with an operator sequence, and have ribosome binding site
sequences and the like, for initiating and completing transcription and translation.
Expression of proteins in prokaryotes is most often carried out in E. coli with
vectors containing constitutive or inducible promoters directing the expression of either
fusion or non-fusion proteins. Fusion vectors add a number of amino acids to an antibody
encoded therein, often to the constant region of the recombinant antibody, without
affecting specificity or antigen recognition of the antibody. Addition of the amino acids

of the fusion peptide can add additional function to the antibody, for example as a marker
(e.g.. epitope tag such as myc or flag).
Other microbes, such as yeast, are also useful for expression. Saccharomyces is a
preferred yeast host, with suitable vectors having expression control sequences (e.g.,
promoters), an origin of replication, termination sequences, and the like as desired.
Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes.
Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase,
isocytochrome C, and enzymes responsible for maltose and galactose utilization.
The vectors containing the polynucleotide sequences of interest (e.g., the heavy
and light chain encoding sequences and expression control sequences) can be transferred
into the host cell by well-known methods, which vary depending on the type of cellular
host. For example, calcium chloride transfection is commonly utilized for prokaryotic
cells, whereas calcium phosphate treatment, electroporation, lipofection, biolistics or
viral-based transfection may be used for other cellular hosts. (See generally, Sambrook et
ah, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd ed., 1989),
incorporated by reference herein in its entirety for all purposes.). Other methods used to
transform mammalian cells include the use of polybrene, protoplast fusion, liposomes,
electroporation, and microinjection (see generally, Sambrook et al., supra). For
production of transgenic animals, transgenes can be microinjected into fertilized oocytes,
or can be incorporated into the genome of embryonic stem cells, and the nuclei of such
cells transferred into enucleated oocytes.
When heavy and light chains are cloned on separate expression vectors, the
vectors are co-transfected to obtain expression and assembly of intact immunoglobulins,
unce expressed, the whole antibodies, their dimers, individual light and heavy chains, or
other immunoglobulin forms of the present invention can be separated as described herein
and/or further purified according to procedures known in the art, including ammonium
sulfate precipitation, affinity columns, column chromatography, HPLC purification, gel
electrophoresis and the like (see generally Scopes, Protein Purification (Springer-Verlag,
N.Y., (1982)). Substantially pure immunoglobulins of at least about 90 to 95%
homogeneity are preferred, and 98 to 99% or more homogeneity most preferred, for
pharmaceutical uses.

Protein Purification
It is desirable to isolate and/or purify proteins expressed according to the present
invention. In certain embodiments, an expressed protein is secreted into the medium and
thus cells and other solids maybe removed, as by centrifugation or filtering for example,
as a first step in the purification process.
In some embodiments, an expressed protein is bound to the surface of the host
cell. In such embodiments, the media is removed and the host cells expressing the
polypeptide or protein are lysed as a first step in the purification process. Lysis of
mammalian host cells can be achieved by any number of means known to those of
ordinary skill in the art, including physical disruption by glass beads and exposure to high
pH conditions.
A protein may be isolated and purified by standard methods including, but not
limited to, chromatography (e.g., ion exchange, affinity, size exclusion, and
hydroxyapatite chromatography), gel filtration, centrifugation, or differential solubility,
ethanol precipitation or by any other available technique for the purification of proteins
(See, e.g., Scopes, Protein Purification Principles and Practice 2nd Edition, Springer-
Verlag, New York, 1987; Higgins, S.J. and Hames, B.D. (eds.), Protein Expression : A
Practical Approach, Oxford Univ Press, 1999; and Deutscher, M.P., Simon, M.I.,
Abelson, J.N. (eds.), Guide to Protein Purification : Methods in Enzymology (Methods in
Enzymology Series, Vol 182), Academic Press, 1997, each of which is incorporated
herein by reference in its entirety). For immunoaffinity chromatography in particular, the
protein may be isolated by binding it to an affinity column comprising antibodies that
were raised against that protein and were affixed to a stationary support. Affinity tags
such as an influenza coat sequence, poly-histidine, or glutathione-S-transferase can be
attached to the protein by standard recombinant techniques to allow for easy purification
by passage over the appropriate affinity column. Protease inhibitors such as phenyl
methyl sulfonyl fluoride (PMSF), Ieupeptin, pepstatin or aprotinin may be added at any or
all stages in order to reduce or eliminate degradation of the polypeptide or protein during
the purification process. Protease inhibitors are particularly advantageous when cells
must be lysed in order to isolate and purify the expressed polypeptide or protein.
Proteins expressed according to certain methods of the present invention may
have more extensive and/or modified glycosylation patterns than they would if grown
under non-inventive cell culture conditions. Thus, one practical benefit of the present
invention that may be exploited at the purification step is that the additional and/or

modified sugar residues present on a glycoprotein grown in accordance with certain of the
present inventive methods and/or compositions may confer on it distinct biochemical
properties that may be used by the practitioner to purify that glycoprotein more easily, or
to a greater purity, than would be possible for a glycoprotein grown in accordance with
non-inventive methods and/or compositions.
One of ordinary skill in the art will appreciate that the exact purification technique
may vary depending on the character of the polypeptide or protein to be purified, the
character of the cells from which the polypeptide or protein is expressed, and/or the
composition of the medium in which the cells were grown.
Pharmaceutical Formulations
The protein preparations of the invention can be formulated as pharmaceutical
compositions in the presence of a pharmaceutical^ acceptable carrier or excipient.
Compositions containing the protein preparations, as described herein, may be
administered to a subject or may first be formulated for delivery by any available route
including, but not limited to parenteral, intravenous, intramuscular, intradermal,
subcutaneous, oral, buccal, sublingual, nasal, bronchial, opthalmic, transdermal (topical),
transmucosal, rectal, and vaginal routes. Inventive pharmaceutical compositions typically
include a purified polypeptide or protein expressed from a mammalian cell line, a
delivery agent (i.e., a cationic polymer, peptide molecular transporter, surfactant, etc., as
described above) in combination with a pharmaceutical^ acceptable carrier. As used
herein, the language "pharmaceutically acceptable carrier" includes solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying
agents, and the like, compatible with pharmaceutical administration. Supplementary
active compounds can also be incorporated into compositions of the present invention.
For example, a protein or polypeptide produced according to the present invention may
be conjugated to drugs for systemic pharmacotherapy, such as toxins, low-molecular-
weight cytotoxic drugs, biological response modifiers, and radionuclides (see e.g., Kurtz
et al, Calicheamicin derivative-carrier conjugates, US 04/0082764 Al). Additional
ingredients useful in preparing pharmaceutical compositions in accordance with the
present invention include, for example, flavoring agents, lubricants, solubilizers,
suspending agents, fillers, glidants, compression aids, binders, tablet-disintegrating
agents, encapsulating materials, emulsifiers, buffers, preservatives, sweeteners,

thickening agents, coloring agents, viscosity regulators, stabilizers or osmo-regulators, or
combinations thereof.
Alternatively or additionally, a protein or polypeptide produced according to the
present invention may be administered in combination with (whether simultaneously or
sequentially) one or more additional pharmaceutically active agents. An exemplary list
of these pharmaceutically active agents can be found in the Physicians' Desk Reference,
55 Edition, published by Medical Economics Co., Inc., Montvale, NJ, 2001, incorporated
herein by reference. For many of these listed agents, pharmaceutically effective dosages
and regimens are known in the art; many are presented in the Physicians' Desk Reference
itself.
Solid pharmaceutical compositions may contain one or more solid carriers, and
optionally one or more other additives such as flavoring agents, lubricants, solubilizers,
suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrating
agents or an encapsulating material. Suitable solid carriers include, for example, calcium
phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose,
methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine, low melting
waxes or ion exchange resins, or combinations thereof. In powder pharmaceutical
compositions, the carrier may be a finely divided solid which is in admixture with the
finely divided active ingredient. In tablets, the active ingredient is generally mixed with a
carrier having the necessary compression properties in suitable proportions, and
optionally, other additives, and compacted into the desired shape and size.
Liquid pharmaceutical compositions may contain the polypeptide or protein
expressed according to the present invention and one or more liquid carriers to form
sututions, suspensions, emulsions, syrups, elixirs, or pressurized compositions.
Pharmaceutically acceptable liquid carriers include, for example water, organic solvents,
pharmaceutically acceptable oils or fat, or combinations thereof. The liquid carrier can
contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers,
preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors,
viscosity regulators, stabilizers or osmo-regulators, or combinations thereof. If the liquid
formulation is intended for pediatric use, it is generally desirable to avoid inclusion of, or
limit the amount of, alcohol.
Examples of liquid carriers suitable for oral or parenteral administration include
water (optionally containing additives such as cellulose derivatives such as sodium
carboxymethyl cellulose), alcohols or their derivatives (including monohydric alcohols or

polyhydric alcohols such as glycols) or oils (e.g., fractionated coconut oil and arachis oil),
For parenteral administration the carrier can also be an oily ester such as ethyl oleate and
isopropyl myristate. The liquid carrier for pressurized compositions can be halogenated
hydrocarbons or other pharmaceutically acceptable propellant.
Liquid pharmaceutical compositions which are sterile solutions or suspensions
can be administered parenterally, for example by, intramuscular, intraperitoneal, epidural,
intrathecal, intravenous or subcutaneous injection. Pharmaceutical compositions for oral
or transmucosal administration may be either in liquid or solid composition form.
In certain embodiments, a pharmaceutical composition is formulated to be
compatible with its intended route of administration. Solutions or suspensions used for
parenteral, intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline solution, fixed oils,
polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial
agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or
sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium
chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or
sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use typically include sterile
aqueous solutions (where water soluble) or dispersions and sterile powders for the
extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline, bacteriostatic water,
CREMOPHOR EL'M (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all
cases, the composition should be sterile and should be fluid to the extent that easy
syringability exists. Advantageously, certain pharmaceutical formulations are stable
under the conditions of manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi. In general, the
relevant carrier can be a solvent or dispersion medium containing, for example, water,
ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol,
and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of surfactants. Prevention of the
action of microorganisms can be achieved by various antibacterial and antifungal agents,

for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In
certain cases, it will be useful to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, or sodium chloride in the composition. Prolonged
absorption of the injectable compositions can be brought about by including in the
composition an agent which delays absorption, for example, aluminum monostearate and
gelatin.
Sterile injectable solutions can be prepared by incorporating the purified
polypeptide or protein in the required amount in an appropriate solvent with one or a
combination of ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating the purified
polypeptide or protein expressed from a mammalian cell line into a sterile vehicle which
contains a basic dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the preparation of sterile injectable
solutions, advantageous methods of preparation are vacuum drying and freeze-drying
which yields a powder of the active ingredient plus any additional desired ingredient from
a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. For the
purpose of oral therapeutic administration, the purified polypeptide or protein can be
incorporated with excipients and used in the form of tablets, troches, or capsules, e.g.,
gelatin capsules. Oral compositions can also be prepared using a fluid carrier, e.g., for
use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets, pills, capsules, troches
and the like can contain any of the following ingredients, or compounds of a similar
nature: a cinder such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel,
or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as
colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring
agent such as peppermint, methyl salicylate, or orange flavoring. Such preparations may
be mixed chewable or liquid formulations or food materials or liquids if desirable, for
example to facilitate administration to children, to individuals whose ability to swallow
tablets is compromised, or to animals. Formulations for oral delivery may
advantageously incorporate agents to improve stability within the gastrointestinal tract
and/or to enhance absorption.

For administration by inhalation, inventive compositions comprising a protein
preparation expressed from a mammalian cell line and a delivery agent can also be
administered intranasally or by inhalation and are conveniently delivered in the form of a
dry powder inhaler or an aerosol spray presentation from a pressurised container, pump,
spray, atomiser or nebuliser, with or without the use of a suitable propellant, e.g.
"dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a
hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A™) or 1,1,1,2,3,3,3-
heptafluoropropane (HFA 227EA™), carbon dioxide or other suitable gas. In the case of
a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a
metered, for example a therapeutically effective amount. The present invention
particularly contemplates delivery of inventive compositions using a nasal spray, inhaler,
or other direct delivery to the upper and/or lower airway. Intranasal administration of
DNA vaccines directed against influenza viruses has been shown to induce CD8 T cell
responses, indicating that at least some cells in the respiratory tract can take up DNA
when delivered by this route, and inventive delivery agents will enhance cellular uptake.
According to certain embodiments, compositions comprising a purified polypeptide
expressed from a mammalian cell line and a delivery agent are formulated as large porous
particles for aerosol administration.
Modified release and pulsatile release oral dosage forms may contain excipients
that act as release rate modifiers, these being coated on and/or included in the body of the
device. Release rate modifiers include, but are not exclusively limited to,
hydroxypropylmethyl cellulose, methyl cellulose, sodium carboxymethylcellulose, ethyl
cellulose, cellulose acetate, polyethylene oxide, Xanthan gum, Carbomer, ammonio
metnacryiate copolymer, hydrogenated castor oil, carnauba wax, paraffin wax, cellulose
acetate phthalate, hydroxypropylmethyl cellulose phthalate, methacrylic acid copolymer
and mixtures thereof. Modified release and pulsatile release oral dosage forms may
contain one or a combination of release rate modifying excipients. Release rate
modifying excipients may be present both within the dosage form i.e., within the matrix,
and/or on the dosage form, i.e., upon the surface or coating.
Systemic administration can also be by transmucosal or transdermal means. For
transmucosal or transdermal administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration, detergents, bile salts, and
fusidic acid derivatives. Transmucosal administration can be accomplished through the

use of nasal sprays or suppositories. For transdermal administration, the purified
polypeptide or protein and delivery agents can be formulated as a suitable ointment
containing the active compound suspended or dissolved in, for example, a mixture with
one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene
glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
Alteenatively, They can be formulated as a suitable lotion or cream, suspended or
dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan
monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax,
cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
Alternatively, the compounds can be administered in the form of a suppository or
pessary, or they may be applied topically in the form of a gel, hydrogel, lotion or other
glycerides, solution, cream, ointment or dusting powder.
In some embodiments, compositions are prepared with carriers that will protect
the protein against rapid elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery systems. In general,
inventive compositions may be formulated for immediate, delayed, modified, sustained,
pulsed, or controlled-release delivery. Biodegradable, biocompatible polymers can be
used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,
polyorthoesters, and polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. Suitable materials can also be obtained
commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions (including liposomes targeted to infected cells with monoclonal antibodies to
viral antigens) can also be used as pharmaceutically acceptable carriers. These can be
prepared according to methods known to those skilled in the art, for example, as
described in U.S. Patent No. 4,522,811.
Proteins produced according to the present invention may also be used in
combination with a cyclodextrin. Cyclodextrins are known to form inclusion and non-
inclusion complexes with certain molecules. Formation of a cyclodextrin complex may
modify the solubility, dissolution rate, bioavailability and/or stability property of a
protein or polypeptide. Cyclodextrin complexes are generally useful for most dosage
forms and administration routes. As an alternative to direct complexation with the
protein or polypeptide, the cyclodextrin may be used as an auxiliary additive, e.g. as a
carrier, diluent or solubiliser. Alpha-, beta- and gamma-cyclodextrins are most

commonly used and suitable examples are described in published international patent
applications WO91/11172, WO94/02518 and WO98/55148.
In some embodiments, pharmaceutical compositions of the present invention are
provided in unit dosage form, such as tablets or capsules. It may be advantageous to
formulate oral or parenteral compositions in unit dosage form for ease of administration and uniformity ofdosage. In such form, the composition is sub-divided in unit dose
containing appropriate quantities of the polypeptide or protein. The unit dosage forms
can be packaged compositions, for example packeted powders, vials, ampoules, pre-filled
syringes or sachets containing liquids. The unit dosage form can be, for example, a
capsule or tablet itself, or it can be an appropriate number of any such compositions in
package form. As one skilled in the art will recognize, therapeutically effective unit
dosage will depend on several factors, including, for example, the method of
administration, the potency of the polypeptide or protein, and/or the weight of the
recipient and the identities of other components in the pharmaceutical composition.
A protein preparation, e.g., pharmaceutical composition containing the same, can
be administered at various intervals and over different periods of time as required, e.g.,
one time per week for between about 1 to 10 weeks, between 2 to 8 weeks, between about
3 to 7 weeks, about 4, 5, or 6 weeks, etc. The skilled artisan will appreciate that certain
factors can influence the dosage and timing required to effectively treat a subject,
including but not limited to the severity of the disease or disorder, previous treatments,
the general health and/or age of the subject, and other diseases present. Treatment of a
subject with a polypeptide or protein as described herein may comprise a single treatment
or a series of treatments. It is furthermore understood that appropriate doses may depend
upon the potency of the polypeptide or protein and may optionally be tailored to the
particular recipient, for example, through administration of increasing doses until a
preselected desired response is achieved. It is understood that the specific dose level for
any particular animal subject may depend upon a variety of factors including the activity
of the specific polypeptide or protein employed, the age, body weight, general health,
gender, and diet of the subject, the time of administration, the route of administration, the
rate of excretion, any drug combination, and the degree of expression or activity to be
modulated.
The present invention encompasses the use of the compositions described herein
for treatment of nonhuman animals. Accordingly, doses and methods of administration
may be selected in accordance with known principles of veterinary pharmacology and

medicine. Guidance may be found, for example, in Adams, R. (ed.), Veterinary
Pharmacology and Therapeutics, 8th edition, Iowa State University Press; ISBN:
0813817439; 2001.
The pharmaceutical compositions can be included in a container, pack, or
dispenser together with instructions for administration.
The following examples are illustrative and not intended to be limiting.
Examples
Solutions of the Antibodies M1-M3 were evaluated by static light scattering,
dynamic light scattering and asymmetric flow field flow fractionation at different salt
concentrations, i.e., different ionic strength.
Antibody Solution Preparation
All solutions were prepared by dialysis into appropriate salt-containing buffer
solution at room temperature, overnight with two buffer exchanges.
Instrumental Methods:
Static Light Scattering
Static light scattering (SEC-MALS) was used to measure the weight average
molecular weights of the antibody solutions. For the static light scattering experiments,
the antibody solutions were diluted to 4 mg/mL and multiple samples were manually
injected into the instrument. The antibody solutions were measured at 20°C using static
light scattering at multiple angles (45, 90 and 135) using a Mini Dawn instrument from
Wyatt Technology. The scatter data at 90° was used to calculate the weight average
molecular weight. The complete data sets (multiple angles) were used to create Zimm
plots that were used to calculate molecular weight and second virial coefficients.
Dynamic Light Scattering
Dynamic light scattering was used to measure the particle size of the antibodies in
solution. The antibody solutions were measured at 90° angle at 25°C using a DynaPro
instrument from Wyatt Technology.

Asymmetric Flow Field Flow Fractionation
Asymmetric Flow Field Flow Fractionation (AF4) was also used to measure the
particle size of the antibodies in solutions. AF4 measurements were performed on an
Eclipse instrument from Wyatt Technology. AF4 analysis parameters were as follows:
temperature 20C, running buffer is the same as the dialysis buffer, 10KDa cutoff
membrane.
Optical Density
Turbidity of a protein sample was measured as an apparent optical density
(absorbance of light at a specific wavelength) using a SpectraMax UV-Vis at a
wavelength of 400nm.
Example 1: Analysis of IgGl Antibody Ml (Myo-029)
Antibody Ml solutions exhibit a large opalescence effect depending on salt
concentration, i.e., ionic strength. Figure 1 shows the visually perceptible change in
opalescence as concentrations increase from 0 mM NaCl to 150 mM NaCl at
approximately 20mg/mL of protein. Antibody Ml solutions ranging from 0 mM NaCl to
100 mM NaCl were also analyzed by dynamic light scattering, samples were diluted to
approximately 5 mg/mL. The data collected are shown in Table 1. Figure 1 shows
images of Antibody Ml (also referred to herein as "Myo-029") solutions exposed to
increasing NaCl concentration (showing a visually perceptible opalescence increase).
Figure 2 shows the dynamic light scattering plots for the 0 mM NaCl solution and
the lOOmM NaCl Antibody Ml solutions the data for which is included in Table 1.


The increase in % mass of the Peak 2 and Peak 3 species correlates with the
increase in opalescence that is visually perceptible in Figure 1. The presence of Peak 3 in
the 0 mM NaCl solution indicates that a small amount of the opalescent species is present
even at low salt concentrations for Antibody Ml.
Asymmetric flow field flow fractionation of a 100 mM NaCl solution of Antibody
M1 (at 20mg/mL protein) indicated a large species as shown in Figure 3. SEC-HPLC
(samples diluted to 3mg/mL) was unable to discern any large species for a similar 100
mM NaCl solution of Antibody Ml as shown in Figure 4.
The reversibility of the opalescence effect for Antibody Ml was also examined.
An Antibody M1 solution was dialyzed from 0 mM NaCl to 100 mM NaCl and then back
to 0 mM NaCl. Antibody Ml was dialyzed overnight into a 100 mM NaCl solution
(originally in a salt free solution), then a small amount of this material at 100mM NaCl
was dialyzed overnight back into a salt-free solution. Figure 5 shows the visually
perceptible change in opalescence for these three Antibody Ml solutions. Figure 6 shows
dynamic light scattering plots for the opalescent 100 mM NaCl intermediate dialysis
solution and also the 0 mM NaCl final dialysis solution.
These data indicate that Antibody Ml contains an extremely large species, though
the species is only present in a relatively small amount at 0 mM NaCl, and that the
concentration of this large species increases with increasing salt concentration. The data
also show that the large species can be discerned using asymmetric flow field flow
fractionation, but is too large to make it onto an SEC-HPLC column, is disrupted by the
bedding, or is disrupted upon dilution. Fortunately, the large species can be resolved and
analyzed using dynamic light scattering as shown.
Example 2: Analysis of IgG 1 Antibody M2
Antibody M2 solutions do not exhibit a large opalescence effect over the
concentration ranges relevant to subcutaneous antibody dosing. Figure 7 shows the lack
of a visually perceptible change in opalescence as concentrations increase from 0 mM
NaCl to 150 mM NaCl at approximately 20mg/mL of protein. Antibody M2 solutions of
0 mM NaCl and 150 mM NaCl were also analyzed by dynamic light scattering at
approximately 5mg/mL of protein. The dynamic light scattering data collected are shown
in Table 2. Figure 8 shows the dynamic light scattering plots for the 0 mM NaCl solution
and the 150mM NaCl Antibody M2 solutions the data for which is included in Table 2.


These data show that Antibody M2 does not have an appreciable amount of an
opalescent species in the concentration range relevant to subcutaneous dosing. In fact,
the Peak 3 percent mass only rises at the 150 mM NaCl level to the percent mass level for
Antibody M at 0 mM NaCl.
Example 3: Analysis of Antibody M3
Antibody M3 solutions do not exhibit a large opalescence effect over the
concentration ranges evaluated. Antibody M3 solutions of 0 mM NaCl and 150 mM
NaCl were analyzed by dynamic light scattering at approximately 5mg/mL. The dynamic
light scattering data collected are shown in
Table 3.
Figure 9 shows the dynamic light scattering plots for the 0 mM NaCl solution and
the 150mM NaCl Antibody M3 solutions, the data for which are included in
Table 3.

These data show that Antibody M3 does not have an appreciable amount of an
opalescent species in the concentration range evaluated. In fact, the Peak 3 percent mass
only rises at the 150 mM NaCl level to a percent mass level just higher than that for
Antibody Ml at 0 mM NaCl.

Example 4: Evaluation of Second Virial Coefficients
Static light scattering was used to determine the molecular weights and second
virial coefficients (A2) for the antibodies analyzed in Examples 1-3 (Antibody Ml,
Antibody M2, and Antibody M3). Second virial coefficient measurements can be
determined using static light scattering and Zimm analysis. The experiment is conducted
by injecting; multiple-dilute protein samples manually into a light scattering system.
Using these multiple measurements and precise concentrations, a Zimm plot can be
constructed. These data are shown in Table 4.
Table 4: Molecular Weight and Second Virial Coefficient Data Calculated
from Static Light Scattering Data

The second virial coefficient data show the aggregation/association tendencies of
Antibody M1, i.e., negative second virial coefficients, as compared to Antibody M2 and
Antibody M3, which have positive virial coefficients. Figure 10 shows a second virial
coefficient plot for Antibody Ml at 100mM NaCl and Figure 11 shows a second virial
coefficient plot for Antibody M2 at 150 mM NaCl from a concentration range of 0.1 to
1.1 mg/mL of protein. These data indicate that the second virial coefficient can be used
as a predictor of opalescence for a particular antibody.
Example 5: Effects of Salt Identity and Concentration on Opalescence
in Antibody Ml
To evaluate the effects of salt identity on the opalescence of Antibody Ml, several
experiments were conducted.

Opalescence change with salt identity over time:
Four 63 mg/mL Antibody Ml solutions were prepared: a control solution with no
salt, a solution with 100 mM NaHPO4, a solution with 100 mM NaCl, and a solution with
100 mM CaCl2. These solutions were then allowed to sit at room temperature for one
hour. Images of these solutions after one hour are shown in Figure 12. As can be seen in
Figure 12, the order of opalescence after one hour is NaCl > NaHPO4 > CaCl2. This
image also shows that opalescence is not solely related to the presence of chloride ions,
i.e., the NaHPO4 solution developed opalescence.
These same solutions were then kept at 2-8°C for two weeks. Images of these
solutions after two weeks are shown in Figure 13. Each of these solutions shown in Fig.
13 showed gelling after two weeks at 2-8°C and developed opalescence. However, the
degree of gelling and opalescence was salt type and concentration dependent. The order
of opalescence was the same as after one hour: NaCl > NaHPO4 > CaCl2. Also tested,
but not shown at this time, was the salt MgCl2, which fit into the scheme as follows:
NaCl >NaHPO4 > MgCl2 > CaCl2.
Opalescence change with salt identity and temperature cycling:
Changes in optical density at 400 run was used to monitor the effect of salt
identity on opalescence during temperature cycling. For this experiment, four 40 mg/mL
Antibody Ml solutions were prepared: a control solution with no salt, a solution with 100
mM NaHPO4, a solution with 100 mM NaCl, and a solution with 100 mM CaCl2. These
solutions were cycled from 2-8°C to room temperature for three cycles. Each cycle from
2-8°C to room temperature and back to 2-8°C took 24 hours. A plot of OD400 nm versus
number of cycles is shown in Figure 14. The increasing OD400 nm values for the NaCl
solution, for example, indicates the increasing formation of secondary structure. Thus,
the data in Figure 14 shows that cycling between liquid and gel likely increases
opalescence.
Opalescence change with salt identity and concentration:
Percent high molecular weight changes were monitored by SEC-HPLC for
changes in salt concentration. For this experiment, CaCl2, NaCl, MgCl2, and NaHPO4
solutions were prepared at 0, 5, 10, and 20 mM concentrations. The percent high
molecular weight material was measured for each sample. A plot of percent high

molecular weight versus concentration is shown in Figure 15. The percent high
molecular weight species increase for each salt except CaCl2.
Changes in optical density with salt identity and concentration:
Changes in optical density at 400 run were used to monitor the effect of salt
identity and concentration on opalescence. For This experiment, a series of Antibody Ml
solutions were prepared using MgCl2, CaCl2 and NaC1 salts. A plot of OD400 ran versus
salt concentration is shown in Figure 16. Figure 17 shows an expanded region of the data
in Figure 16. These data illustrate that high molecular weight species increases at
increasing concentration of NaCl and only slightly increases for CaCl2 and MgCl2.
Changes in optical density with respect to Antibody Ml concentration in 20 mM
CaCl2:
Finally, changes in optical density at 400 nm were used to monitor the effect of
changing Antibody Ml concentration in a 20 mM CaCl2 solution. For this experiment,
10, 20, 50, 60, and 75 mg/mL solutions of Antibody Ml were prepared in 20 mM CaCl2.
A plot of OD400 nm versus Antibody Ml concentration is shown in Figure 18. These
data show that higher order structure increases with increasing Antibody Ml
concentration.
Several observations can be made regarding all the data for Example 5. The
opalescence effect is salt dependent in the following manner: NaCl > Na2PO4 > MgCl2 >
CaCl2. For a given protein concentration, opalescence will initially increase and then
either plateau or drop as the salt concentration is increased. Opalescence increases with
protein concentation. All the saits evaluated gelled Antibody Ml at 2-8°C and the
timing of gelation was salt dependent and appeared to be related to the level of
opalescence.
The antibody used in the present invention is preferably Antibody Ml (also
referred to herein as "Myo-029"), Antibody M2, and Antibody M3. Where the ionic
strength of an antibody preparation (preferably containing Antibody Ml, Antibody M2,
or Antibody M3) is to be reduced or decreased in accordance with the present invention,
the reduction or decrease in the ionic strength is normally carried out so that ratio of the
ionic strength to the antibody concentration is reduced.
All publications, patent applications, patents, and other references mentioned
herein are incorporated by reference herein in their entirety.

Other embodiments are within the scope of the following claims.

WHAT is CLAIMED is:
1. A method for decreasing the opalescence of an antibody preparation, the
method comprising: modifying the ratio of the ionic strength to the antibody
concentration in the antibody preparation such that the opalescent appearance of the
antibody preparation is reduced, wherein the antibody preparation, prior to modifying
the ionic strength, shows an opalescence greater than about 2 European
Pharmacopoeia standard at a pharmaceutically effective concentration of about 100
mg/ml.
2. A method for reducing the formation of high molecular weight species in
an antibody preparation, the method comprising: modifying the ratio of the ionic
strength to the antibody concentration of in the antibody preparation such that the
high molecular weight species in the protein preparation are reduced.
3. The method of either claim 1 or 2, wherein the antibody preparation
comprises a human, humanized, chimeric, CDR-grafted, or an in vitro generated
antibody, or an antigen-binding fragment thereof.
4. The method of either claim 1 or 2, wherein the antibody preparation
comprises an IgGl or an IgG4 heavy chain constant region.
5. The method of either claim 1 or 2, wherein the antibody preparation
comprises an anti-GDF8 antibody.

6. The method of either claim 1 or 2, wherein the antibody of the antibody
preparation has a negative second virial coefficient in a salt concentration ranging
from about 50 to 200 mM.
7. The method of claim 6, wherein the second virial coefficient of the
antibody is about -1 to -10 x 10-3, or about -1 to -10 x 10-4 mol-mg/g2 in an
antibodypreparation containing 100 mM sodium chloride.

8. The method of either claim 1 or 2, wherein the antibody of the antibody
preparation has a positive virial coefficient in a salt concentration ranging from about
50 to 200 mM.
9. The method of claim 8, wherein the second virial coefficient of the
-antibody is about 1 to 10 x 10-4 or about 1 to 10 x 10-3 mol-mg/g2 in an antibody
preparation containing 100 mM sodium chloride.
10. The method of any one of claims 1 to 9, wherein the antibody is present at
a concentration of about 5 to 300 mg/ml in the antibody preparation, prior to and/or
after practicing the method.
11. The method of any one of claims 1 to 10, wherein the ionic strength of the
antibody preparation is reduced by decreasing the concentration of a salt present in
the preparation.
12. The method of claim 11, wherein the ionic strength of the antibody
preparation is reduced by decreasing the concentration of a salt present in the
preparation, wherein the salt present in the preparation is selected from the group
consisting of sodium chloride, calcium chloride, magnesium chloride and sodium
phosphate.
13. The method of claim 11, wherein the ionic strength of the antibody
preparation is reduced by decreasing the concentration of a salt present in the
preparation, wherein the concentration of the salt is reduced to at least about two-,
three-, five-, ten- or one hundred-fold lower than the concentration in the opalescent
preparation.
14. The method of claim 11, wherein the ionic strength of the antibody
preparation is modified by replacing a first salt with a second salt, wherein the first
salt is NaCl, and the second salt is selected from the group consisting of NaHPO4,
MgCl2, and CaCl2.

15. The method of claim 11, wherein the ionic strength of the antibody
preparation is modified by replacing a first salt with a second salt, wherein the first
salt is NaHPO4, and the second salt is selected from the group consisting of MgCl2
and CaCl2.
16. The method or anyone of claims 1 to 10, wherein the ionic strength of the
antibody preparation is modified by ultra-filtration or dialysis.
17. The method of any one of claims 1 to 10, further comprising:
evaluating the opalescence of the antibody preparation; or
determining the salt concentration in the antibody preparation; wherein
the ionic strength of the antibody preparation is modified by a process comprising
one of more of: applying one or more filtration methods or reducing the salt
concentration of one or more steps in the process of purifying the antibody.
18. The method of claim 17, wherein the process of purifying the antibody
comprises one or more of centrifugation, filtration, chromatography, lyophilization or
reconstitution.
19. The method of any one of claims 1 to 10, further comprising evaluating
the opalescence of the antibody preparation.
20. The method of claim 19, wherein the opalescence of the antibody
preparation is detected by evaluating the turbidity of the solution, evaluating a change
in the second virial coefficient, or evaluating a change in the formation of high
molecular weight species.
21. The method of claim 20, wherein the opalescence is reduced to about 2 or
1 European Pharmacopoeia standard or less.
22. The method of claim 20, wherein the second virial coefficient changes
from about -1 to -10 x 10'3 to about -1 to -10 x 10-4.

23. The method of claim 20, wherein the amount of high molecular weight
species is reduced by about two-, three-, four-, five-, six-, seven-, eight-, nine-, ten-,
fifty- or one hundred-fold.
24: A method of decreasing the formation of high molecular weight species in
an antibody preparation, the method comprising modifying the ratio of the ionic
strength to the antibody concentration in the antibody preparation such that the
formation of high molecular weight species in the protein preparation is reduced,
wherein the antibody in the preparation has a negative virial coefficient in 100 mM
sodium chloride solution.
25. A method of improving the efficiency of a purification process of an
antibody preparation, the method comprising:
(optionally) evaluating whether the protein preparation forms an
opalescent solution at one or more salt concentrations;
modifying the ratio of the ionic strength to the antibody concentration
in the preparation, such that the high molecular weight species in the antibody
preparation is reduced, thereby improving the efficiency of a purification process of
the antibody preparation,
wherein the ionic strength of the solution is modified by one or more of: applying one
or more filtration methods, replacing the salt used in the protein preparation with a
lower opalescent inducer, or reducing the ionic strength of one or more steps in the
26. A method of improving the production of an antibody, the method
comprising:
recovering a first antibody solution; and
evaluating the level of opalescence or presence of high molecular
weight species in the first antibody solution;
wherein (a) if the level of opalescence or high molecular weight species is
equal to or less than a predetermined level, then selecting said first antibody solution
for use in the antibody product; or (b) if the level of opalescence or high molecular

weight species is greater than a predetermined level, then selecting a second antibody
solution having a second ionic strength.
27. The method of claim 26, further comprising repeating the evaluating step
until the level of opalescence in the solution is equal to or less than the predetermined
level.
28. The method of claim 26 or 27, wherein the antibody solution is recovered
by a protein purification method chosen from one or more of: antibody elution from a
matrix, recovery of a dialyzed solution or other filtrate, solubilizing a dry preparation,
or a chromatographic method.
29. The method of claim 28, wherein the antibody solution is recovered by a
chromatographic method and the chromatographic method is selected from the group
consisting of Protein A chromatography, affinity chromatography, hydrophobic
interaction chromatography, immobilized metal affinity chromatography, size
exclusion chromatography, diafiltration, ultrafiltration, viral removal filtration, anion
exchange chromatography, hydroxyapatite chromatography and cation exchange
chromatography.
30. The method of any one of claims 26 to 29, wherein the predetermined
level of the antibody solution has a turbidity value of about 2.
21. A method of determining the propensity of an antibody preparation to
have an opalescent appearance, the method comprising:
(optionally) identifying an antibody as having a tendency to self-
aggregate;
providing one or more samples of the antibody preparation; and
detecting the formation of high molecular weight species upon
increasing the salt concentration in the one or more samples, wherein an increase in
the formation of high molecular weight species upon increasing salt concentration is
indicative of an increased propensity of the antibody in the preparation to have an
opalescent appearance.

32. A method of evaluating the opalescence of an antibody sample, the
method comprising:
providing an antibody sample;
determining the turbidity or presence of high molecular weight species at
one or more salt concentrations; and
providing a report on the level of opalescence in the antibody sample as
a function of turbidity or presence of high molecular weight species at the one or
more salt concentrations.
33. An antibody preparation produced by the method of any of claim 1, 2, 25
or 26.
34. A pharmaceutically-acceptable antibody preparation comprising an
antibody at a concentration of at least about 50 mg/ml in less than about 50 mM NaCl.

Methods for reducing the opalescent appearance of a protein preparation by modifying the ionic strength of the preparation, as well as compositions, e.g., pharmaceutical compositions, of concentrated protein with decreased opalescence are disclosed. Purification methods which monitor and/or reduce the salt concentrations at selected steps are also disclosed.