PREDICTION OF RELATIVE POLYPEPTIDE SOLUBILITY BY
POLYETHYLENE GLYCOL PRECIPITATION
FIELD OF THE INVENTION
[0001] The present invention relates generally to the field of protein characterization.
More specifically, the invention relates to methods of predicting protein solubility.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0002] This application claims priority to provisional U.S. Application Serial
No. 60/801,862, filed on May 19, 2006, which is herein incorporated by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0003] An important aspect of formulating pharmaceutical compositions that contain
a polypeptide is determining the solubility of the polypeptide to be used in a preparation.
Procedures for determining solubility are generally not easily used for evaluating the
solubility of large numbers of polypeptides because, for example, of the number of
manipulations used and difficulties with obtaining large enough quantities of the
polypeptide(s) to be tested.
[0004] Polyethylene glycol (PEG) is a non-toxic, non-adsorbing, synthetic long-chain
amphiphilic polymer that is widely used in a number of industrial applications. PEG is a
useful molecule within a laboratory or industrial setting because it can be used at ambient
temperatures for polypeptide precipitation.
[0005] There is a need for a high throughput screening method to assay the solubility
of polypeptides that are candidates, e.g., as drugs, at an early stage of discovery or
development, and thereby to identify those polypeptides that may possess problematic
solubility at a relatively early stage of development, for example, before commercial
scaling. Additionally, minimizing the amount of starting material required for testing
solubility is advantageous, e.g., when the polypeptide is available only in very limited
amounts.

SUMMARY OF THE INVENTION
[0006] The invention relates to methods for predicting the relative solubility of one or
more polypeptides comprising precipitating the polypeptides using PEG volume
exclusion. The assay is referred to herein as a "relative solubility assay" or "PEG
precipitation assay." More particularly, the test polypeptides assayed by the present
method can be compared to one or more polypeptides of known solubility to detect those
polypeptides with potentially difficult solubility problems prior to the time-consuming
and expensive commercial scale-up of producing the test polypeptide. The method can
also be used to identify parameters suitable for various uses of a selected polypeptide.
[0007] Accordingly, the invention relates to a method for predicting the relative
solubility of a test polypeptide. The method includes providing one or more samples of a
test polypeptide in a solution, thereby providing test samples; contacting the test samples
with different concentrations of polyethylene glycol (PEG) thereby forming a precipitated
sample; determining the precipitation of each test sample contacted with PEG; and
correlating the amount of precipitation of the test polypeptide in the precipitated sample
with solubility of at least one reference polypeptide sample analyzed under corresponding
conditions, thereby determining the solubility of the test polypeptide relative to the
reference polypeptide sample; or correlating the amount of precipitation of the test
polypeptide in the precipitated sample(s) under different experimental conditions, thereby
determining the relative solubility of the test polypeptide under each experimental
condition. In some embodiments, the test polypeptide is an antibody or a fragment of an
antibody, a molecule that can bind to a ligand, or a soluble receptor. In certain
embodiments, the method also includes graphing the log of the solubility values
determined for each sample against the PEG concentration of that sample and
extrapolating the resulting line to zero percent PEG, thereby providing an apparent
solubility value for the polypeptide. In some cases, the test polypeptide does not bind to
PEG. In certain embodiments, the PEG precipitation of a test polypeptide is reversible.
The PEG precipitation may, in some cases, not change the secondary structure of the test
polypeptide. For some embodiments, the starting concentration of the test polypeptide to
be analyzed does not substantially affect the resulting solubility value. The method also
includes embodiments in which increasing the temperature increases the solubility value
for a selected PEG concentration or the addition of sucrose to the buffer increases the

solubility of the test polypeptide. The method also can be practiced such that the slope of
the curve resulting from plotting the log solubility values of a higher molecular weight
polypeptide sample against the PEG concentration increases relative to the slope of the
curve of a lower molecular weight polypeptide. In some embodiments of the invention,
the reference is a polypeptide of known solubility. In some cases, several polypeptides of
known solubility are used as references, e.g.. to establish a standard curve with which the
relative solubility of a test polypeptide can be determined. In certain cases, the reference
polypeptide(s) are selected to be of a similar type to the test polypeptide, for example,
antibodies of known solubility can be used as reference polypeptides when determining
the relative solubility of test polypeptides that are antibodies. In some embodiments,
precipitation is assayed by determining turbidity of the precipitated samples). In some
embodiments, the precipitated sample is centrifuged and the amount of precipitate is
determined, the amount of protein in the supernatant is determined, or the amount of
protein in the precipitate is determined.
[0008] In another aspect, the invention relates to a method for determining the
relative solubility of a polypeptide compared to at least one other polypeptide of
approximately the same molecular weight. The method includes providing a sample of at
least two different polypeptides at the same concentration; contacting each polypeptide
sample with a range of test PEG concentrations; determining the lowest test PEG
concentration that precipitates a polypeptide sample, thereby determining a minimum
percentage of PEG that precipitates each polypeptide; and correlating the minimum
percentage of PEG with the solubility of each polypeptide relative to each other
polypeptide. In some embodiments of the memod, one or more manipulations of the
assay are performed in a 96-well plate format. In some embodiments of the method, the
range of PEG concentrations is about 2%-l 6%. The plate or other multisample format
may be read visually by determining the smallest test concentration of PEG that causes
opalescence of a sample. In some cases, the opalescence of samples in the plate is read
using an automated plate reader.
[0009] Unless otherwise defined, all technical and scientific terms used herein have
the same meaning as commonly understood by one of ordinary skill in the art to which
this invention belongs. Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the present invention, suitable

methods and materials are described below. All publications, patent applications, patents,
and other references mentioned herein are incorporated by reference in their entirety. In
addition, the materials, methods, and examples are illustrative only and not intended to be
limiting.
[0010] Other features and advantages of the invention will be apparent from the
detailed description, drawings, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Fig 1 is a graph depicting the results of a binding study of P1 with PEG-1 OK
by Fourier Transform Infrared Spectrometry (FTIR).
[0012] Fig. 2 is a graph depicting the results of a secondary structure analysis of PI
by FTIR.
[0013] Fig. 3 is a bar graph depicting the results of an experiment designed to test
whether polypeptide precipitation with PEG is fully reversible.
[0014] Fig. 4 is a graph depicting the results of experiments comparing the accuracy
of solubility prediction by PEG-1 OK and PEG-20K. Solubility was tested using PEG-
10K and PEG-20K to compare the effectiveness of volume-exclusion methodology with
alternative molecular weight PEG.
[0015] Fig. 5A is a graph depicting the results of experiments in which polypeptides
with different molecular weights were used to test the effect of polypeptide size on the
phase diagram.
[0016] Fig. 5B is a graph depicting the relationship between molecular weight of a
polypeptide and the slope of the line in a graph (as in Figs. 1 and 2) representing
solubility versus PEG precipitation percentage.
[0017] Fig. 6A is a graph indicating the reproducibility of polypeptide solubility
prediction for P4. The experiments were performed in triplicate. 20 mM succinate is the
formulation buffer for P4.
[0018] Fig. 6B is a graph indicating the reproducibility of polypeptide solubility
prediction for P1. The experiments were performed in triplicate. 50 mM histidine is the
formulation buffer for P1.

[0019] Fig. 7 A is a graph depicting the effect of polypeptide concentration on the
PEG-determined solubility of P4 in 20 mM succinate pH6.0. Initial polypeptide
concentrations are 5.5 mg/mL (squares) and 11 mg/mL (diamonds).
[0020] Fig. 7B is a graph depicting the effect of polypeptide concentration on the
PEG-determined solubility of P1 in 20 mM succinate pH6.0. Initial polypeptide
concentrations are 5.5 mg/mL (squares) and 11 mg/mL (diamonds).
[0021] Fig. 8 A is a graph depicting the effect of variable temperature (diamonds,
20°C or squares, 0°C) on predicted solubility of P4 in 20 mM succinate, pH 6.0.
[0022] Fig. 8B is a graph depicting the effect of variable temperature (diamonds,
20°C or triangles, 0°C) on predicted solubility of P1 in 20 mM succinate, pH 6.0.
[0023] Fig. 9 is a graph illustrating the effect of pH (triangles, 20 mM succinate, pH
6.0; squares, 10 mM phosphate, pH 7.0; diamonds 10 mM Tris, pH 8.0) on solubility
estimation of P1.
[0024] Fig. 10 is a graph of the pH profile of P1 solubility predicted by PEG-10K at
0°C and 20°C.
[0025] Fig. 11 is a graph illustrating the effect of the ionic strength of the buffer on
the performance of PEG precipitation method using P1 at 10 mg/mL.
[0026] Fig. 12A is a graph depicting the results of experiments assaying the effect of
sucrose on P5 apparent solubility with NaCl added to the PEG-precipitation buffer.
[0027] Fig. 12B is a graph depicting the results of experiments assaying the effect of
sucrose on P5 apparent solubility without NaCl added to the PEG-precipitation buffer.
[0028] Fig. 13 is a reproduction of a photograph of 96-well plates used in high
throughput screening (HTS) to determine the apparent solubility of monoclonal antibody
using a PEG precipitation method.
[0029] Fig. 14 is a graph depicting the correlation of opalescence of monoclonal
antibody solutions at a concentration of 90 mg/mL with relative solubility predicted by
the PEG precipitation method.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The methods disclosed herein provide advantages for evaluation of
polypeptide characteristics, e.g., solubility. The methods assay the relative solubilities of
polypeptides such as antibodies or fragments of antibodies, using a limited number of

manipulations. Limiting the number of manipulations is an advantage, for example,
because it can reduce the amount of time to obtain a solubility measurement for a
polypeptide or group of polypeptides, and because fewer manipulations minimizes the
amount of polypeptide lost in processing.
Relative Solubility Assay
[0031] The invention relates to the need for a relatively rapid and efficient method for
estimating the relative solubility of a polypeptide (a relative solubility assay). In general,
the method employs PEG precipitation in a method for assaying relative solubility, which
can decrease the amount of starting polypeptide for a solubility assay from approximately
200 mg in conventional approaches that measure actual solubility using a membrane-
based concentration approach, to about 10 mg to about 30 mg (e.g., about 5 mg to about
100 mg, about 5 mg to about 50 mg, or about 10 mg to about 50 mg). The assay method
does not preclude the use of larger amounts of polypeptide.
[0032] In some embodiments, the assay includes adding selected concentrations of
PEG (a PEG precipitation series) to test samples containing a polypeptide of interest in
solution (test polypeptide; a selected protein), determining the saturation concentration of
the polypeptide at each PEG concentration, and comparing the extrapolated value of the
saturation line at zero PEG concentration with at least one additional (i.e., different)
polypeptide tested under the same assay conditions. In other embodiments, a test
polypeptide is prepared under two or more different conditions such as different buffer
components, pH, or temperature and tested for solubility with varying PEG
concentrations. Saturated concentration, which is obtained by measuring polypeptide
concentration in the supernatant of samples in which precipitation is observed, can be
plotted in log scale against corresponding PEG concentration. The Y-intercept of the
fitted line provides the apparent solubility of the polypeptide at zero PEG, and the slope
of the line can be also calculated. Although the apparent solubility can be very different
from actual achievable solubility determined using a membrane-based concentration
approach, the apparent solubility can be utilized to compare relative solubility of one
polypeptide to another. The slope of the fitted line is related to the molecular sizes of
PEG and polypeptide, while it is unrelated to pH, temperature, and buffer.

[0033] In one embodiment, the invention provides a method for predicting the relative
solubility of a polypeptide (e.g., a test polypeptide), the method comprising providing at
least one sample of a test polypeptide in a solution, contacting each sample of the test
polypeptide with a different concentration of polyethylene glycol (PEG), determining the
relative solubility (e.g., by testing the amount of precipitation) of each sample at a given
PEG concentration, and comparing the solubility of the test polypeptide to the solubility
of a reference polypeptide sample or second test polypeptide sample analyzed under
corresponding conditions, thereby determining the relative solubility of the test
polypeptide compared to the reference or second test polypeptide. Additional test
polypeptides may be tested for relative solubility, e.g., three, four, five, ten, twenty, fifty,
one hundred, one thousand, or more, using the method. In some cases, the relative
solubility of multiple samples of the test polypeptide prepared or tested under different
experimental conditions is compared, thereby determining the solubility of the test
polypeptide relative to the second polypeptide or set of experimental conditions. In
certain embodiments, the polypeptides are proteins, e.g., antibodies, antibody fragments,
ligand-binding molecules, or soluble receptors. More than one type of polypeptide can be
used in an assay or the assay may utilize polypeptides that are all of the same or similar
type, e.g., all antibodies.
[0034] The invention further relates to a method as described herein that also includes
graphing the log of the solubility values determined for each sample against the PEG
concentration of that sample and extrapolating the resulting line to zero percent PEG,
thereby providing an apparent solubility value for a given polypeptide sample, or a set of
solubility values for the tested polypeptides. In some aspects of the method, the
polypeptide does not bind to PEG, the PEG precipitation is reversible, the PEG does not
change the secondary structure of the polypeptide, or the starting concentration of the
polypeptide to be analyzed does not substantially affect the resulting solubility value.
Further aspects of the method include increasing the temperature to increase the solubility
value for a given (selected) PEG concentration, or adding sucrose to the buffer to affect
(e.g., increase) the solubility of the polypeptide.
[0035] In still another aspect, the method for predicting the relative solubility of a
polypeptide is performed and analyzed such that the slope of the curve resulting from
plotting the log solubility values of a higher molecular weight polypeptide sample against

the PEG concentration increases relative to the slope of the curve of a lower molecular
weight polypeptide sample.
[0036] In another embodiment, the method provided herein can also include
providing multiple polypeptide samples of different polypeptides at the same
concentration and each different polypeptide is mixed with a range of PEG
concentrations, the minimum percentage of PEG (that is, the minimum percentage of a
tested PEG concentration) that precipitates each different polypeptide is determined (the
minimum precipitating PEG concentration, MPPC, which can be expressed as a
percentage or concentration), and MPPC is correlated with the solubility of the
polypeptide relative to the other polypeptide samples.
[0037] In some embodiments, the polypeptide samples used in a method described
herein are analyzed in a 96-well plate format. In general, the range of PEG
concentrations is about 2-16%. The plate can be read visually by determining the
smallest (lowest) concentration of PEG that results in visible opalescence in the sample
well or the opalescence of sample wells in the plate can be read using an automated plate
reader or other suitable device.
Solubility Assay of Variable Parameters
[0038] In some embodiments, the PEG assay for determining relative solubility of a
polypeptide is used to assay the relative solubility of a selected polypeptide under
different assay conditions, i.e., using different parameters that can affect solubility. This
type of assay is useful, for example, to identify parameters under which the solubility of a
polypeptide is appropriate for a particular purpose such as storage and use as a clinical
compound.
[0039] One example of a parameter that can be varied in the assay is buffer
composition. Buffers that can be tested include, but are not limited to, succinate,
histidine, or phosphate buffers. In some cases, testing relative solubility of a polypeptide
in the presence of different buffers is useful for identifying an appropriate buffer for a
particular application of the polypeptide.
[0040] Density of a solution containing a polypeptide can also affect solubility.
Accordingly, a parameter that can be tested using the assay is effect of varying
concentrations of a molecule that can affect density or other properties of a solution on

solubility. An example of such a molecule is sucrose. Concentrations of sucrose that can
be used in the assay are, for example, about 0.5%-10%. Other molecules that are
relatively inert and can affect the density of a solution can also be used, for example,
dextran or glycerol.
[0041] Another parameter that can be assayed for the effect on relative solubility of a
polypeptide is varying ionic strength. Non-limiting examples of ionic strength that can be
tested include such cations as Na+, Ca2+, K+, Co2+, Cu2+, Fe2+, Mg2+, Ni2+, Zn2+ ,Al3+,
Fe3+, or such anions as Cl-1, NO3-, PO43-, SO42-, CO32-, or C2H3O2- (acetate).
[0042] An additional parameter that can be varied in assays of relative solubility is
temperature (e.g., from about 0°C to about 30°C, about 5°C to about 40°C, about 5°C to
about 37°C, about 15°C to about 37°C, or about 25°C to about 37°C). Another parameter
that can be varied and tested in the assay is pH (e.g., from about pH 5.0 to about pH 8.5;
about pH 5.5 to about pH 8.0; about pH 5.5 to about 7.5, and about pH 6.0 to about pH
7.5).
[0043] Suitable concentrations of polypeptides used in the assay include, without
limitation, about 1 mg/mL to about 200 mg/mL.
[0044] As used herein, "actual solubility" of a polypeptide refers to the maximum
amount of polypeptide that can be dissolved into a solution, the measurement of which
takes place in the absence of a volume-exclusion agent such as PEG. Specific conditions
are, for example, temperature, buffer, ionic strength, pH, solution density, or a
combination thereof.
[0045] As used herein, "relative solubility" of a polypeptide refers to the solubility of
one polypeptide (generally, a test polypeptide) compared to a second polypeptide or
group of polypeptides, or, in some cases, the solubility of a polypeptide under one set of
conditions (parameters) compared to the same polypeptide under one or more different
conditions. Unlike actual solubility, relative solubility does not have a numerical value,
but rather is used to make comparisons, such as with reference polypeptide standards of
known solubility or relative solubility of a polypeptide under different conditions such as
buffer, ionic strength, pH, solution density, or a combination of variations of such
conditions.
(0046] As used herein, "apparent solubility" or "predicted solubility" of a polypeptide
is the numeric value calculated by extrapolating the curve generated on a graph when log

solubility values are plotted against the PEG concentration of a polypeptide sample, the
extrapolation being to the axis representing log solubility and representing the data point
corresponding to a polypeptide solubility when the PEG concentration of the polypeptide
sample is zero.
[0047] The apparent solubility value can include a component reflecting the
interactions of the polypeptide with itself in solution. This is referred to as an "activity
term" and may inflate the apparent solubility value obtained by extrapolating the line
taken from volume-exclusion assays, rendering the apparent solubility value inaccurately
high. This is generally the case for polypeptides with relatively high solubility, such as
albumin, which has a maximum actual solubility of 677 mg/mL based on the packing
density of hexagonally close-packed hard spheres. However, in PEG precipitation
experiments, that number may appear much higher owing to the inclusion of the activity
term in the apparent solubility. The methods disclosed herein for determining relative
solubility do not provide an accurate calculation of actual solubility, but do provide
methods for comparing the solubility of polypeptides to each other under the same
conditions or the same polypeptide to itself under different experimental conditions.
[0048] In one example of an application of a relative solubility assay, a polypeptide or
polypeptide of unknown solubility is compared to a polypeptide known to have low
solubility, e.g., the P5 antibody in the Examples. A protein or polypeptide having
solubility similar to a poorly soluble polypeptide will also have low solubility. Such
information is useful for determining, e.g., appropriate conditions for applications using
such a protein or polypeptide, or can be used to screen out a protein or polypeptide for
applications where low solubility is not acceptable. Thus, a relative solubility assay can
be used to identify polypeptides that are likely to cause similar solubility problems in
large-scale production if the results of the PEG-precipitation method for the two
polypeptides are very similar, or if the test polypeptide shows a lower relative solubility
than the polypeptide of known low solubility.
Precipitation of Polypeptides
[0049] The relative solubility assay disclosed herein includes PEG precipitation of
one or more selected (e.g., test) polypeptides (e.g., at least two selected polypeptides, at
least three selected polypeptides, at least five selected polypeptides, at least ten selected

polypeptides, or more than ten polypeptides). The number of polypeptides that can be
tested in a single assay is generally limited by the available format (e.g., multi-well plate
or printed grid) and the ability to carry out the steps for the number of polypeptides within
an reasonable time. PEG precipitation is carried out by adding a solution of PEG to an
aqueous solution containing the selected polypeptide, resulting in a PEG/polypeptide
solution; incubating the PEG/polypeptide solution for a time sufficient to permit
precipitation of polypeptide in the solution, typically 30-60 minutes. Different times can
be used and may be determined empirically using methods that will be apparent to those
in the art. The assay components (including the polypeptide and PEG) are typically
mixed, e.g.. by pipetting or shaking, at room temperature and incubated at the desired
temperature until time sufficient for measurement of the precipitate has elapsed, typically
about 30-60 minutes. Precipitated polypeptide can be removed (e.g., by centrifugation)
and the amount of polypeptide remaining in the supernatant or in the precipitate is
determined, and solubility for that polypeptide is calculated. Alternatively, instead of the
collecting of precipitate, precipitation is assayed, e.g., by assaying the opalescence (e.g.,
turbidity) of the PEG/polypeptide solution. In some cases, precipitation is assayed by
determining the amount of precipitate collected by centrifugation or determining the
amount of protein in the collected precipitate.
[0050] Methods of assaying opalescence are known in the art and include, for
example, assaying absorbance at a wavelength of 400 nm or higher by UV/visible
spectrophotometer, other methods of photo-electric turbidometry (e.g., automated
turbidometry), simple visualization by eye, right angle light scattering, or fluorescence.
Examples of PEG suitable for use in a relative solubility assay includes, without
limitation, PEG-1 OK, PEG-20K, or within a range of approximately PEG 4-30K. In
general, ultrapure PEG is used although other qualities of PEG preparation can be suitable
(e.g., chemical grade, commercial grade, or pharmaceutical grade).
Polypeptides
[0051] The methods described herein are generally used for testing the relative
solubility of polypeptides including polypeptide fragments. However, the method can be
used to test the relative solubility of any type of molecule that can be precipitated using
PEG. In general, a polypeptide that is tested for relative solubility using the methods

described herein is an isolated or purified protein or polypeptide. Such molecules are
generally substantially free of cellular material or other contaminating polypeptides from
the cell or tissue source from which the protein or polypeptide is derived, or, when the
molecule to be tested is chemically synthesized, the sample containing the molecule is
substantially free from chemical precursors or other chemicals. The language
"substantially free" means preparation of a selected protein or polypeptide having less
than about 30%, 20%, 10%, or 5% (by dry weight), of a protein or polypeptide that is not
the selected protein or polypeptide (also referred to herein as a "contaminating
polypeptide"), or of chemical precursors. When the selected protein or polypeptide is
produced by recombinant means, it is also generally substantially free of culture medium,
i.e.. culture medium represents less than about 20%, less than about 10%, and less than
about 5% of the volume of the protein or polypeptide preparation.
[0052] "Polypeptide" as used herein means a chain of amino acids regardless of
length or post-translational modifications, and includes, for example, proteins, peptides,
protein or polypeptide fragments, and conjugated proteins. The term also includes
polypeptides that contain non-naturally-occurring amino acids. Polypeptides can be
obtained from any source, for example, secreted recombinant polypeptides, polypeptides
isolated from natural sources, non-secreted recombinant polypeptides, or synthetic
polypeptides. Polypeptide concentrations suitable for use in the assay are from about 0.S
mg/mL to 10 mg/mL, about 10 mg/mL to 100 mg/mL, and about 100 mg/mL to 300
mg/mL. Proteins used in an assay can be denatured or have secondary or tertiary
structures (e.g., naturally occurring structure or structure induced during, for example,
isolation. If impurities in the sample are substantially less soluble than the peptide of
interest, the apparent solubility will be under estimated. In contrast, if the impurities are
substantially more soluble than the peptide of interest, the apparent solubility of the
peptide of interest will be overestimated.
Determination of Relative Solubility
[0053] To determine the relative solubility of a polypeptide or set of polypeptides, the
turbidity or other measure of precipitation (such as protein content of a precipitate or of a
supernatant following PEG precipitation of a sample) can be plotted against the variable
(e.g., PEG concentration, pH, ionic strength, buffer molarity, sucrose concentration, or a

combination thereof). For example, the Y-intercept of a selected polypeptide or set of
polypeptides is compared to the Y-intercept of one or more polypeptides assayed under
the same conditions and the solubilities of the polypeptides are ranked (e.g., less soluble
to more soluble), thereby providing a measure of relative solubility. Other methods of
determining relative solubility are described herein, and include visual evaluation of
opalescence and correlation of such evaluation with relative solubility.
Validation of the method
[0054] The relative solubility assay was validated by comparing the predicted
outcomes of changes in experimental parameters such as:
(i) temperature, which increased the solubility of polypeptide(s),
(ii) starting polypeptide concentration, which did not affect the measurements of
relative solubility at a concentration range of about 1 mg/mL to about 100 mg/mL,
(iii) pH, which increased solubility as pH decreased from pH 8.0 to pH 6.0,
(iv) ionic strength of buffer, which reduced solubility as ionic strength was increased,
and also was compensated for by the addition of salt (NaCl), and
(v) sucrose, which improved solubility, even of polypeptides having relatively low
solubility.
[0055] All of these results were consistent with findings related to varying parameters
and solubility using methods known in the art. Therefore, the relative solubility assay can
be used to provide useful information about the solubility of a polypeptide that is
consistent with solubility determined by other methods.
[0056] Thus, the results of the relative solubility assay disclosed herein are consistent
with predicted outcomes when assay conditions are varied, suggesting further that the
PEG precipitation method of determining relative solubility is a suitable substitute for
actual solubility determinations, which may require tenfold greater amounts of starting
polypeptide.
High Throughput Screening (HTS) Using a Relative Solubility Assay
[0057] The relative solubility assay described herein can be used in a method for
large scale analysis of selected polypeptides by employing a 96-well format or other

format designed to accommodate multiple samples (e.g, in wells or printed grids) for
simultaneous analysis.
[0058] In an example of such an assay, different polypeptides with similar molecular
weights (such as different antibodies, which will have the same slope of the line in the
solubility graph if the molecular weights are approximately equal) are suspended at the
same polypeptide concentration and are mixed with a range of PEG concentrations (e.g.,
about 1-20%) in a 96-well or other multi-well format such as a slide printed with a
hydrophobic grid, incubated for a sufficient time for precipitation to occur, and visually
screened for the lowest PEG concentration that precipitates each polypeptide. The lowest
PEG concentration is then correlated with the approximate relative solubility of the
polypeptide.
[0059] The format allows analysis of multiple polypeptide samples relative to one
another by determining the approximate concentration of PEG at which a polypeptide
begins to precipitate, as assayed by observation of which samples are becoming visibly
clouded or opaque (e.g., assaying turbidity). This technique can thus omit the need for
centrifugation of the precipitate and obtaining a concentration reading on the supernatant
as in other techniques. However, in some cases of the present method, such methods
(e.g., centrifugation and concentration readings) can also be used.
[0060] To analyze the results of a high-throughput assay for relative solubility (e.g.,
an assay used to screen a set of polypeptides for relative solubility), turbidity can be
visually screened (by examining the opalescence in the sample wells), or alternatively,
automate the process using a UV/visible spectrophotometer with measurements in the
400-600 nm range, for example, at 500 nm.
[0061] As used herein, the term "opalescence" means detectable turbidity or other
visual indication that a polypeptide solution (e.g., a PEG/polypeptide solution) contains a
precipitate. In some cases, opalescence is not detectable to the human eye. In such cases,
analysis of samples, e.g., the high-throughput screening samples, can be determined using
more sensitive methods such as spectrophotometry, e.g., automated spectrophotometry,
by using a visible light spectrophotometer or equivalent means for detecting light
absorbance of the samples.

EXAMPLES
[0062] The invention is further illustrated by the following examples. The examples
are provided for illustrative purposes only. They are not to be construed as limiting the
scope or content of the invention in any way.
Example 1. General Methodology for Performing PEG-Precipitation of Polypeptides
[0063] All PEG used in the experiments described infra was purchased from Fluka
Chemical Corp. (Ronkonkoma, NY). Dissolving PEG in buffered solutions was observed
to cause a significant change in the measured pH; as much as 1 pH unit with 40% PEG-
10K in 20 mM succinate buffer. This pH change could change the slope of the solubility
curve by progressively increasing the pH with increasing PEG concentration. Therefore,
the pH values of the 40% PEG-10K stock solutions were adjusted after dissolving PEG in
a buffer.
[0064] Antibody stock solutions were prepared by dialyzing the polypeptide into a
selected buffer and diluted to 10 mg/mL or 5 mg/mL with a buffer. Aliquots of the
polypeptide solution and 40% PEG-10K solution were added to 1.5 mL Eppendorf tubes
to a final volume of 350 µl according to the Table 1, and thoroughly mixed.


[0065] All solutions were allowed to equilibrate at a target temperature for at least
30 minutes. Precipitation was observed to occur at certain polypeptide to PEG ratios. All
mixtures were centrifuged to separate the polypeptide precipitate, and the supernatant
assayed by ultraviolet and visible spectrophotometry at 280 nm and 320 nm. The
temperature of the samples was maintained at 20°C or 0°C (in an ice water bath)
throughout the incubation and centrifugation process. An ice water bath at 0°C was
chosen to reduce temperature fluctuation because of the high heat capacity of water at
0°C. A solubility diagram was plotted and fitted by exponential function using the
saturation solubility data in the log linear scale as a function of PEG concentration.
Example 2. Assay for Polypeptide-PEG Interactions
[0066] To examine whether polypeptides of interest (selected polypeptides) interacted
with PEG, and therefore would interact with PEG in a relative solubility assay, which
would adversely affect the analysis of the assay results, a binding study was performed.
Two small columns were prepared with 0.5 mL of MabSelect™ ProA resin (GE
Healthcare, Piscataway, NJ) loaded into each column. Both columns were washed with
10 mL 10 mM phosphate pH 7.0 to remove ethanol. Two mL of 30 mg/mL of an
antibody (P1) in the same buffer was added to each of the columns, and the flow-through
was reloaded onto the column to insure maximum binding. Each column was then
washed with 10 mL of binding buffer (10 mM phosphate pH 7.0) to remove unbound
polypeptide. Twenty percent PEG-10K in 50 mM histidine pH 6.0 was then added to one
of the columns followed by a wash using 10 mL of the same buffer. The resin in each
column was suspended in 1 mL of water and each suspension transferred to a 10 mL
lyophilization vial. The samples in each of the two vials were lyophilized. The following
three samples were analyzed using Fourier Transform Infrared Spectroscopy (FTIR):
PEG-10K powder, lyophilized ProA-mAb resin incubated with PEG, and lyophilized
ProA-mAb resin not incubated with PEG. Three mg of each powder sample was mixed
with 200 mg of KBr, pressed into a 13-mm disk at four tons pressure with a die press.
Fourier Transform Infrared Spectroscopy (FTIR) analysis of the KBr pellets was
conducted with an MB FTIR spectrometer (ABB Bomen Inc., Quebec, Canada). FTIR is
an analytical technique that is used to identify organic materials by measuring the
absorption of various infrared light wavelengths by the polypeptide. The absorption of

infrared light creates bands of absorption, which are characteristic of specific molecular
components and structures. A total of 256 scans at 2 cm-1 resolution were averaged to
obtain each spectrum. During data acquisition, the spectrometer was continuously purged
with dry air to eliminate the spectral contribution of atmospheric water. As the results in
Fig. 1 indicate, PEG does not bind to P1. While conjugated polypeptides are
contemplated for testing using a relative solubility assay, a molecule conjugated to a
selected polypeptide generally must not interact with PEG. The method described in this
Example can be modified using methods known in the art to test for PEG interaction with
a molecule.
Example 3. Assay for Structural Changes in the Polypeptide
[0067] To determine whether any structural changes in the polypeptide takes place
during the PEG precipitation protocol, aqueous P1 antibody not contacted with PEG was
analyzed in parallel with P1 antibody precipitated by the PEG technique. Ten mg/mL P1
in 50 mM histidine pH 6.0 was precipitated by adding 40% PEG solution to a final PEG
concentration of 12%, and the precipitate was collected by centrifugation. The
precipitated polypeptide and PI solution at 30 mg/mL were loaded into a BioCell liquid
cell (Biotools, Inc., Wauconda, IL) equipped with CaF2 windows, and measured by ABB
Bomen MB FTIR spectrometer. The spectra were corrected for water contribution,
smoothed with a 9-point smoothing function, normalized, and analyzed by second
derivatization in the amide I region. As shown in Fig. 2, PEG precipitation of the
polypeptide did not induce a change in the secondary structure of the polypeptide. This
result was consistent with expectations based on knowledge in the art and thus confirms
that the PEG precipitation method is useful for determining the relative solubility of a
polypeptide.
Example 4. Analysis of the Reversibility of the PEG Precipitation Method
(0068] Validation of the PEG precipitation method (relative solubility assay) requires
that the volume-exclusion curve generated by measuring polypeptide content in the
supernatant following precipitation results from equilibrium between soluble and
precipitated polypeptide. Equilibrium indicates that there is no net change between solid
and aqueous phases of the polypeptide in the reaction, and depends on the solid phase

being capable of returning to the aqueous phase ("reversibility"). To test the reversibility
of the disclosed method, PEG-precipitated P1 antibody was re-dissolved and the
supernatant was re-quantified to compare with the amount of starting polypeptide. One
mL of 10 mg/mL PI antibody in 50 mM histidine pH 6.0 was precipitated by adding 40%
PEG-10K in the same buffer to a final PEG concentration of 14%. Supernatant
concentration was measured using a UV-visible spectrophotometer. Two mL of 50 mM
histidine pH 6.0 was then added to the mixture to fully dissolve the precipitate,
centrifuged, and the concentration of polypeptide in the supernatant was measured. The
amount of total soluble polypeptide was calculated by multiplying the concentration and
the volume. As shown in the data of Fig. 3, the amount of P1 antibody recovered after
being re-dissolved is not significantly less than the starting amount, indicating the method
is fully reversible, demonstrating mat this assay requirement is met.
Example 5. Effect of PEG Molecular Weight
(0069] To compare the effectiveness of volume-exclusion with different molecular
weights of PEG, solubility was tested using PEGr-10K and PEG-20K (Fig. 4). P1
suspended in 50 mM histidine buffer, pH 6.0 was used as a starting polypeptide, and the
experiment was carried out at 20°C. Precipitation of 10 mg/mL P1 requires a slightly
lower concentration of PEG-20K (about 7% and above) compared to PEG-10K
concentration (about 8.5% and above) because PEG-20K has higher efficiency of protein
precipitation.
[0070] Both types of PEG resulted in a similar Y-intercept despite the difference in
the slope, indicating that both PEG types give similar apparent solubility values. The
high viscosity of PEG-20K stock solution made it difficult to handle during sample
preparation; therefore, PEG-10K was chosen for subsequent studies.
Example 6. Effect of Molecular Weight of the Polypeptide
[0071] Additional polypeptides with different molecular weights were used to test the
effect of polypeptide size on the solubility measurements using the relative solubility
assay. Fig. 5 A discloses the resulting curves of each polypeptide tested. The slopes of
the respective lines for each polypeptide were then plotted against the molecular weight

of the polypeptide, and the resulting graph (Fig. 5B) indicates that the slope increases as
the polypeptide size increases.
[0072] Some noticeable features when using the method of PEG-induced polypeptide
precipitation can be understood with reference to Fig. 4. The apparent solubility values of
4679 mg/mL and 5223 mg/mL, which are estimated by the intercept, are inaccurately
high- It is reported that the estimated maximum solubility of albumin is 677 mg/mL, i.e.,
it is sterically impossible to pack much more than 667 mg of protein into 1 mL of volume
based on the packing density of hexagonally close-packed hard spheres (Atha and
Ingham, J. Biol. Chem. 256:12108-12117 (1981)). Atha and Ingham point out that
polypeptides at high concentration result in intercepts that include an activity related
term, and therefore exceed the practical solubility limits. Consequently, care should be
taken in interpretation of data for highly soluble polypeptides. The extrapolated apparent
solubility does not depict the actual solubility. Thus, the PEG precipitation method
should be considered qualitative rather than quantitative in the following experiments,
i.e., the method can be used to compare one polypeptide to another rather than using the
method to determine with accuracy the actual solubility of a single polypeptide.
Example 7. Reproducibility of the Relative Solubility Assay
[0073] Two different monoclonal antibodies, P4 (in 20 mM succinate pH 6.0) and P1
(in 50 mM histidine pH 6.0) were both used to test the reproducibility of the relative
solubility assay using the protocols described in Example 1. Solubility measurements
were carried out in triplicate runs on different days (Figs. 6A and 6B). At both
temperatures, good reproducibility of solubility prediction was observed for both
monoclonal antibodies. Thus, the method disclosed herein yields reproducible results for
polypeptide solubility of the same polypeptide tested multiple times. This reproducibility
was also observed when PEG precipitation was carried out at different temperatures (i.e.,
the initial temperature was tested twice and yielded consistent results, and the second
temperature was tested twice and produced consistent results). These results indicate that
mere is no significant inter-assay variability in solubility determinations using the PEG
precipitation method. This feature is important for an assay such as the relative solubility
assay that is intended for, e.g., commercial use.

Example 8. Effect of Starting Polypeptide Concentration
[0074] To determine whether the PEG-precipitation method described here remained
independent of polypeptide concentration, P4 antibody and P1 antibody were both tested
at a low concentration of 5.5 mg/mL and a high concentration of 11 mg/mL using the
protocol of Example 1. The effect of varying the total polypeptide content of the solution
on the predicted solubility of the polypeptide is illustrated in Figs. 7A and 7B. For both
tested antibodies, the extrapolated solubility values are independent of the total
polypeptide concentration between 5.5 mg/mL and 11 mg/mL.
|0075] These data demonstrate that the PEG precipitation method for determining
solubility can be used over a range of protein concentrations.
Example 9. Effect of Temperature
[0076] To test whether the PEG-precipitation method for determining relative
solubility accords with the known effect of temperature on solubility of polypeptides, P4
and P1 were both tested using the general protocol of Example 1 but at two different
temperatures: 0°C and 20°C. Increased apparent solubility was found at elevated
temperature (Figs. 8A and 8B) using this approach. A similar temperature effect on
solubility has been found empirically through experimentation, e.g., using a method
testing actual solubility. Thus, the PEG precipitation method described herein is
consistent with the results expected using methods testing actual solubility.
Example 10. Effect of pH
[0077] The solubility of P1 at various pHs was tested (Fig. 9). The log-linear
response of P1 concentration versus percent PEG concentration shows that the Y-
intercept (zero PEG concentration, i.e., apparent solubility) decreases as pH increases
from pH 6 to pH 8, but the slopes are not different. The pH profile (Fig. 10) correlates
well with the expectation that a polypeptide has lowest solubility at pH around its p1 (7.5-
8.0 for P1).
[0078] These data further demonstrate that the PEG precipitation method can produce
results consistent with other methods, such as those for determining actual solubility.

Example 11. Effect of Buffer and Ionic Strength
[0079] Apparent solubility values for P1 antibody were tested at pH 6.0 using
different buffers such as succinate, histidine, and phosphate, and different results were
obtained with various buffers (Fig. 11). These data demonstrate that the low ionic
strength of 10 mM histidine buffer is the explanation for the lack of precipitation that
occurred for 10 mg/mL P1 in that buffer, which could subsequently be compensated for
by the addition of NaCl. Therefore, when performing a relative solubility assay, an
increase in tonic strength can decrease the solubility of a protein. This is in accordance
with expected measurements of actual solubility. This further validates the method as
concurring with results obtained with standard solubility assays known in the art.
Example 12. Effect of Sucrose
[0080] Previous studies have shown that sucrose enhances solubility of PS during
ultrafiltration/diafiltration. To confirm the reliability of the relative solubility assay
method, the effect of sucrose on predicted solubility of P5 was tested (Figs. 12A and
12B). P5 in 10 mM histidine buffer at pH 6.0, 20°C was compared with or without the
addition of 2% sucrose. The results of these experiments are shown in Fig. 12B, and
indicate that the predicted solubility of P5 increased with the presence of sucrose in both
buffers tested.
[0081] The magnitude of sucrose-induced solubility enhancement is generally higher
in buffer with low ionic strength. This was tested in a relative solubility assay by adding
5 mM NaCl to both the sucrose and non-sucrose samples. As indicated in Fig. 12A, NaCl
greatly decreased the sucrose-induced improvement in solubility. These results of a
relative solubility assay agree well with the previous experimentally determined effect of
sucrose on solubility, further validating the relative solubility approach.
Example 13. Employing a Relative Solubility Assay in High Throughput Screening
(HTS)
[0082] A 96-well plate format for high throughput screening was used in a
demonstration of an application of a relative solubility assay in a higher throughput
format using a selection of monoclonal antibodies. Because the slope of the phase
diagram remained constant for different monoclonal antibodies under all different

conditions (buffer, temperature, concentration) tested above, a simplified version of HTS
was designed for this study. All monoclonal antibodies were dialyzed in 50 mM histidine
pH 6.0 and their concentrations were adjusted to 10 mg/mL. Forty percent PEG-10K
stock solution was prepared in the same buffer and pH was adjusted to 6.0. A quartz 96-
well plate was prepared by filling wells with different ratios of monoclonal antibody to
PEG-10K stock solution according to Table 2 to give a final volume of 200 µl in each
well. Each row was designated for a specific monoclonal with increased final PEG
concentration from 2% in column #1 to 16% in column #12. All samples were mixed by
pipetting up and down five times, followed by incubation at room temperature for 15
minutes.
[0083] When the initial polypeptide concentrations of all monoclonal were adjusted to
the same level, more soluble monoclonal antibodies required a higher percentage of PEG
to precipitate. Therefore, the minimum percentage of PEG needed for polypeptide
precipitation indicates relative solubility of the polypeptide (Fig. 13). This simplified
version of the method avoids centrifugation, dilution and concentration measurement of
the supernatant following the precipitation step, resulting in high efficiency and reduced
need for polypeptide material.


[0084] The relationship of the opalescence of the samples (indicating precipitation) of
monoclonal antibodies at 90 mg/mL was measured by spectrophotometer absorbance on a
SPECTRAmax Plus384 Microplate Spectrophotometer (Molecular Devices Corp.,
Sunnyvale, CA) at 500 nm (A500), with the resulting relationship with the relative
solubility (i.e., the lowest PEG concentration at which precipitation was observed) plotted
on the graph in Fig. 14. These results indicate that the opalescence increases as the
relative solubility decreases.
OTHER EMBODIMENTS
[0085] It is to be understood that while the invention has been described in
conjunction with the detailed description thereof, the foregoing description is intended to
illustrate and not limit the scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are within the scope of
the following claims.

CLAIMS
What is claimed is:
1. A method for predicting the relative solubility of a test polypeptide, the method
comprising;
a. providing one or more samples of a test polypeptide in a solution, thereby
providing test samples;
b. contacting the test samples with different concentrations of polyethylene glycol
(PEG), thereby forming a precipitated sample;
c. determining the precipitation of each test sample contacted with PEG; and
d. correlating the amount of precipitation of the test polypeptide in the
precipitated sample with solubility of at least one reference polypeptide sample analyzed
under corresponding conditions, thereby determining the solubility of the test polypeptide
relative to the reference polypeptide sample; or correlating the amount of precipitation of
the test polypeptide in the precipitated sample under different experimental conditions,
thereby determining the relative solubility of the test polypeptide under each experimental
condition.
2. The method of claim 1, wherein the test polypeptide is an antibody.
3. The method of claim 1, wherein the test polypeptide is a molecule that can bind to a
ligand.
4. The method of claim 1, wherein the test polypeptide is a soluble receptor.
5. The method of claim 1, wherein the test polypeptide is an antibody fragment.
6. The method of claim 1, further comprising graphing the log of the solubility values
determined for each sample against the PEG concentration of that sample and
extrapolating the resulting line to zero percent PEG, thereby providing an apparent
solubility value for the polypeptide.
7. The method of claim 1, wherein the test polypeptide does not bind to PEG.

8. The method of claim 1, wherein the PEG precipitation is reversible.
9. The method of claim 1, wherein the PEG does not change the secondary structure of
the test polypeptide.
10. The method of claim 1, wherein the starting concentration of the test polypeptide to
be analyzed does not substantially affect the resulting solubility value.
11. The method of claim 1, wherein increasing the temperature increases the solubility
value for a selected PEG concentration.
12. The method of claim 1, wherein the addition of sucrose to the buffer increases the
solubility of the test polypeptide.
13. The method of claim 1, wherein the slope of the curve resulting from plotting the log
solubility values of a higher molecular weight polypeptide sample against the PEG
concentration increases relative to the slope of the curve of a lower molecular weight
polypeptide.
14. The method of claim 1, wherein the reference is a polypeptide of known solubility.
15. The method of claim 1, wherein precipitation is assayed by determining turbidity.
16. The method of claim 1, wherein the precipitated sample is centrifuged and the
amount of precipitate is determined, the amount of protein in the supernatant is
determined, or the amount of protein in the precipitate is determined.
17. A method for determining the relative solubility of a polypeptide compared to at least
one other polypeptide of approximately the same molecular weight, the method
comprising:

a. providing a sample of at least two different polypeptides at the same
concentration;
b. contacting each polypeptide sample with a range of test PEG
concentrations;
c. determining the lowest test PEG concentration that precipitates a
polypeptide sample, thereby determining a minimum percentage of PEG
that precipitates each polypeptide; and
d. correlating the minimum percentage of PEG with the solubility of each
polypeptide relative to each other polypeptide.
18. The method of 17, wherein at least (b) to (c) are performed in a 96-well plate format.
19. The method of 17, wherein the range of PEG concentrations is about 2%-16%.
20. The method of 17, wherein the plate is read visually by determining the smallest test
concentration of PEG that causes opalescence of a sample.
21. The method of 17, wherein the opalescence of samples in the plate is read using an
automated plate reader.

A method is described for predicting the relative solubility of a polypeptide using polyethylene glycol (PEG) based
volume exclusion precipitation. Different polypeptides can be tested for their solubilities relative to each other or relative to a
reference. A single polypeptide can be tested for its relative solubility under different experimental conditions. The solubility determinations
can be made by comparison based on graphs plotting the log solubility of the polypeptide against a range of PEG
concentrations. Additionally, a method is provided for the high throughput visual or automated screening of multiple polypeptides
for relative solubility differences, in a method that can omit the step of measuring the actual solubility or actual amount of precipitation
of each sample at each PEG concentration.