RESEARCH ARTICLES

Biotechnology
Applications of Differential Scanning Calorimetry for Thermal
Stability Analysis of Proteins: Qualification of DSC
JIE WEN,1 KELLY ARTHUR,2 LETHA CHEMMALIL,3 SALMAN MUZAMMIL,4 JOHN GABRIELSON,2 YIJIA JIANG1
1
Product Attribute Sciences, Amgen Inc., Thousand Oaks, California 91320
2
Analytical Sciences, Amgen Inc., Longmont, Colorado 80503
3
Process Development, Amgen Inc., West Greenwich, Rhode Island 02817
4
Biophysics & Developability, Antibody Drug Discovery, Centocor Inc. Radnor, Pennsylvania 19406

Received 10 August 2011; revised 3 October 2011; accepted 28 October 2011

Published online 6 December 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22820

ABSTRACT: Differential scanning calorimetry (DSC) has been used to characterize protein
thermal stability, overall conformation, and domain folding integrity by the biopharmaceutical
industry. Recently, there have been increased requests from regulatory agencies for the qual-
ification of characterization methods including DSC. Understanding the method precision can
help determine what differences between samples are significant and also establish the accep-
tance criteria for comparability and other characterization studies. In this study, we identify the
parameters for the qualification of DSC for thermal stability analysis of proteins. We use these
parameters to assess the precision and sensitivity of DSC and demonstrate that DSC is suitable
for protein thermal stability analysis for these purposes. Several molecules from different struc-
tural families were studied. The experiments and data analyses were performed by different
analysts using different instruments at different sites. The results show that the (apparent)
thermal transition midpoint (Tm ) values obtained for the same protein by same and different
instruments and/or analysts are quite reproducible, and the profile similarity values obtained
for the same protein from the same instrument are also high. DSC is an appropriate method
for assessing protein thermal stability and conformational changes. 2011 Wiley Periodicals,
Inc. and the American Pharmacists Association J Pharm Sci 101:955964, 2012
Keywords: calorimetry (DSC); thermal analysis; proteins; biotechnology; method
qualification; protein structure

INTRODUCTION continuously measured and calibrated to power units.

Differential scanning calorimetry (DSC) is a thermo-
dynamic technique, which measures heat capacity as
a function of temperature. The signal from a sample
cell is compared with a reference cell lacking protein
in an identical solution environment. As the temper-
ature of the cells is increased, the temperature dif-
ferences between the reference and sample cells are
Abbreviations used: DSC, differential scanning calorimetry;
Tm , thermal transition midpoint or apparent thermal transition
midpoint; Gdn, guanidine hydrogen chloride; CxN: Sodium citrate
with sodium chloride buffer at pH x.
Correspondence to: Jie Wen (Telephone: +805-447-2515;
Fax: +805-375-6416; E-mail: jwen@amgen.com); Yijia Jiang
(Telephone: +805-447-1116; E-mail: yjiang@amgen.com)
Journal of Pharmaceutical Sciences, Vol. 101, 955964 (2012)
2011 Wiley Periodicals, Inc. and the American Pharmacists Association
This data channel is referred to as the DP signal or the
differential power between the reference and sample
cells. The DP signal is converted to heat capacity. The
heat capacity is continuously recorded as a function
of temperature. After buffer subtraction and analysis
of the resulting thermogram, the enthalpy and (ap-
parent) thermal transition midpoints (Tm ) for each
transition can be obtained. By further analysis of the
data, the thermal stability and structural integrity of
the protein can be studied.13
Differential scanning calorimetry has been widely
used to study protein thermodynamics, folding, and
interactions.13 It has also been used to charac-
terize protein thermal stability, overall conforma-
tion, and domain folding integrity during formulation
and product development by the biopharmaceutical

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 3, MARCH 2012 955

956 WEN ET AL.
industry.47 However, until recently no formal evalu-
ation of the method precision has been carried out.
Understanding the method precision can help deter-
mine what differences between samples are signif-
icant and also establish the acceptance criteria for
comparability and other characterization studies.
To assess the precision of DSC for thermal sta-
bility analysis, five proteins from different struc-
tural families were studied using three different
instrument models: the MicroCal VP-DSC, the Micro-
Cal VP-Capillary DSC (MicroCal LLC, Northampton,
Massachusetts), and the TA Instruments Q2000 DSC
(TA Instruments, New Castle, Delaware). Replicate
measurements using the same experimental parame-
ters and procedures were carried out for each protein
at the specified concentrations in the corresponding
buffer. The experiments were performed by different
analysts at different sites on different dates. Intra-
day versus interday precision as well as the effect of
protein concentration were also evaluated.
During downstream processing, low pH buffers
are commonly used for elution and viral inactivation
of proteins produced using mammalian cell lines.8
Guanidine hydrogen chloride (Gdn) is commonly used
to solubilize inclusion bodies of Escherichia coli ex-
pressed protein and for denaturation of proteins.9
During these processes, both low pH and Gdn can
alter thermal stability. To determine if DSC is an ap-
propriate technique to assess protein thermal stabil-
ity changes that can occur under these manufacturing
process conditions, the sensitivity of the DSC tech-
nique was evaluated using several approaches includ-
ing analyses of the effect of pH and denaturant per-
turbation on protein thermal stability. After evaluat-
ing the precision and sensitivity, several case studies
are shown in this manuscript to further demonstrate
that DSC is suitable for thermal stability analysis of
proteins.
METHODS AND MATERIALS
Materials
Proteins from different structural families including
an antibody, Fc conjugate, Fc fusion protein, cytokine,
and a small globular protein were used for this study.
The proteins and related information (concentration
and structural family) used in the study are listed in
Table 1. The proteins were produced at Amgen Inc.
(Thousand Oaks, California) and were made at least
98% pure by size-exclusion chromatography.
Methods
Instruments
All of the DSC instruments were maintained with
proper preventative maintenance. The MicroCal DSC
instruments (MicroCal LLC) were calibrated accord-
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 3, MARCH 2012
Table 1. Proteins Used for the Studies
Protein Structural Family
Protein 1 IgG 2
Protein 2 Fc conjugatea
Protein 3 Fc fusion proteinb
Protein 4 Globular protein containing primarily beta
sheet structure (MW < 50 kDa)
Protein 5 Four-helical-bundle cytokine
Protein 6 IgG 1
Protein 7 Fc conjugate
Protein 8 Helical bundle
Protein 9 IgG 2
Protein 10 Globular protein (MW < 50 kDa)
Protein 11 Fc conjugate
Protein 12 Fc conjugate
a A molecule formed when a peptide is fused to the Fc is referred to as
an Fc conjugate here.
b A molecule formed when a protein is fused to the Fc is referred to as
an Fc fusion protein here.
MW, molecular weight.
ing to the standard MicroCal DP signal calibration
and MicroCal temperature calibration procedures. TA
instrument was calibrated with TA instrument cell
resistance and capacitance calibration and the TA in-
strument cell constant and temperature calibration
using indium.
The following parameters were used for MicroCal
VP-DSC and MicroCal VP-Capillary DSC operation
(MicroCal LLC): postcycle thermostat, 4 C; starting
temperature, 4 C; final temperature, 110 C (90 C for
protein 4 and protein 5); scan rate, 60 C/h; prescan,
15 min; postscan, 0 min; filtering period, 10 s; feed-
back mode/gain, none; thermostat control set point,
4 C.
The following parameters were used for TA DSC
operation: postcycle thermostat, 4 C; pan type, high
volume; gas flow setting, 50 mL/min; starting temper-
ature, 4 C; final temperature, 110 C (90 C for protein
4 and protein 5); scan rate, 60 C/h (48 C/h for protein
3); data sampling interval, 1 s/point. The pans used
for the analysis were obtained from TA Instruments
(cat #900825.902). The pans and lids were made of
stainless steel; O-ring was made of Viton.
Precision Study
For experiments using MicroCal differential scanning
calorimeter (MicroCal LLC), the antibody and Fc con-
jugate (or Fc fusion protein) were prepared at ap-
proximately 0.5 mg/mL, and the cytokine and small
globular protein were prepared at approximately
1.0 mg/mL in the native state just prior to the mea-
surements of each protein. The final concentrations of
each sample were measured using a spectrophotome-
ter at 280 nm. The samples for TA DSC were pre-
pared in a similar manner; the concentrations were
prepared at approximately 25 mg/mL.
DOI 10.1002/jps
 APPLICATIONS OF DSC FOR THERMAL STABILITY ANALYSIS OF PROTEINS
Five proteins from different structural families
were studied. Replicate measurements using the
same experimental parameters and procedure were
executed for each protein in the native state at speci-
fied concentrations. The experiments were conducted
by different analysts at different sites on different
dates. Intraday versus interday precision and concen-
tration effect were also evaluated.
Sensitivity Study
Selected proteins were analyzed in their stable stor-
age buffers, C3N buffer, and in the presence of 6 M
Gdn. The profiles of these proteins in low pH buffer
or 6 M Gdn were compared with that of the control
(folded protein in stable storage buffer). The sensitiv-
ity of the DSC method could be assessed by spiking
the denatured species into a solution of native protein
and correlating the profile similarity with the per-
centage of denatured species present in the sample.
The challenge for the experimental mixing approach
is that the unfolding of a protein by low pH or Gdn is
often reversible or partially reversible. Thus, a stan-
dard curve that correlates the changes in DSC profile
with the percentage of a denatured protein at various
mass ratios cannot be generated empirically because
after mixing the samples, it is virtually impossible to
determine what percentage of native protein remains
as well as the state of the unfolded species in the sam-
ples. To address this issue, we carried out a blending
study using two folded proteins of different structures
as surrogates for the native and unfolded forms of
the same protein. The DSC profiles of the two pro-
teins mixed in defined buffers at different mass ratios
were obtained empirically. Computer-simulated pro-
files were obtained by mathematically blending the
profiles of the individual proteins at the same mass
ratios. The profiles obtained empirically were then
compared with the mathematically obtained profiles
at the same mass ratios. This ensures that the em-
pirical mixing and computer mixing approaches are
equivalent and verifies that the computer simulation
of mixing can be used to assess the sensitivity of DSC.
Data Analysis
The values of Tm were obtained by Origin (Origin-
Lab Corp, Northampton, Massachusetts) and TA uni-
versal analysis software (Thermo Electron, Madison,
Wisconsin).1013 The similarity of the DSC profiles
of the replicate measurements was analyzed using a
quantitative comparison method from Thermo Elec-
tron (Madison, Wisconsin) (OMNIC softwareQC
compare function).14,15 The method correlates the
overall profile information in a specified region of both
sample and reference profiles to determine the sim-
ilarity between the two. The result of the analysis
is reported as a value between 0% and 100%, which
indicates how well the sample profile matches the ref-
DOI 10.1002/jps
957
erence profile. A value of 100% indicates an identical
match. This numerical analysis approach was used
for the DSC profile similarity analysis of all studies
described in this paper.
RESULTS AND DISCUSSION
Qualification of DSC: Precision Assessment
Replicate Measurements of the Same Protein Using the
Same Instrument
The precision of DSC was first assessed by replicate
measurements of the same protein using the same
instrument. Proteins from different structural fami-
lies were selected for analysis. Specifically, protein 1
[immunoglobulin G (IgG) 2], protein 2 (Fc conjugate),
protein 3 (Fc fusion protein), protein 4 (small glob-
ular protein containing primarily beta sheet struc-
ture), and protein 5 (four-helical-bundle cytokine)
were evaluated. The Tm values were obtained by Ori-
gin and TA universal analysis software. The similar-
ity values were obtained by OMNIC softwareQC
compare function (Thermo Electron).14,15 The overlay
of the DSC scans of replicate measurements of pro-
tein 2 from the same instrument, shown in Figure 1,
is representative of the data obtained. There are two
apparent endothermic thermal transitions at approx-
imately 57.4 C and 77.9 C corresponding to the un-
folding of the CH 2 and CH 3 domains. The DSC profiles
of protein 2 are typical of an Fc conjugate, with the two
transitions demonstrating that the protein is properly
folded into the distinct domains. The standard devia-
tions (SDs) of this set of replicate measurements for
each transition are 0.1 C and 0.02 C, respectively, in-
dicating that the DSC method is quite reproducible.
Protein 2 is an Fc conjugate. The unfolding of the
CH 2 and CH 3 domains of an Fc conjugate is often well
resolved. However, for monoclonal antibodies (mAbs),
the unfolding of the Fab domain may overlap (or even
distort) that of the CH 2 and/or CH 3 domain. Therefore,
the DSC profiles of mAbs usually show overlapped
peaks. The typical unfolding DSC profiles of mAbs
can be found in Ref. 7.
The summary of Tm values of replicate measure-
ments of the same protein using the same instrument
is shown in Table 2, and the summary of profile sim-
ilarity values of replicate measurements of the same
protein using the same instrument is shown in Ta-
ble 3. There are two apparent endothermic thermal
transitions observed for proteins 1, 2, and 4, three ap-
parent endothermic thermal transitions for protein 3,
and one for protein 5. The Tm values are shown in sep-
arate columns according to the individual transitions.
The similarity values are listed for the comparison of
the overall profile. The corresponding SDs of the repli-
cate measurements are also listed in Tables 2 (indi-
vidual transitions) and 3 (overall profile). The SDs of
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 3, MARCH 2012
958 WEN ET AL.
the Tm values range from 0.01 C to 1.13 C and the
SDs of profile similarity values range from 0.04% to
3.76%, suggesting that the precision of replicate DSC
measurements from the same instrument is very good
regardless of the differences in protein structure fam-
ilies.
Replicate Measurements of the Same Protein
Using Different Instruments
The precision of the DSC analysis was also assessed
by replicate measurements of the same protein using
different instruments. The summary of Tm and sim-
ilarity values of the replicate measurements of the
same protein using different instruments is listed in
Table 4.
The SDs of the Tm values for the same protein range
from 0.38 C to 1.49 C, which is only slightly greater
than those observed using the same instrument. This
suggests that the precision of the Tm values of the
same protein using different instruments is also good.
Compared with the profile similarity values mea-
sured using the same instrument, those using dif-
ferent instruments vary dramatically (Table 4). The
profiles from the TA instrument are significantly dif-
ferent from those of the MicroCal instruments (Micro-
Cal LLC). Even comparing the profiles from the same
manufacturer, MicroCal, the similarity values and
SDs are much poorer than those obtained on the same
instrument. The similarity values range from 44.2%
to 88.9% and the SDs range from 4.4% to 22.8%. Re-
sults suggest that comparison of thermal melting pro-
files from different DSC instruments/manufacturers
should not be used to determine protein confirmation
and stability changes.
Figure 1. Representative overlay of the DSC scans of
protein 2 from the same instrument in formulation buffer.
Black, run 1; red, run 2; blue, run 3. The experiment was
performed on a MicroCal VP-Capillary DSC. Cp is heat
capacity
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 3, MARCH 2012
The thermal transition temperatures are relatively
constant and reproducible regardless of site, instru-
ment, or analyst. The profile similarity values ob-
tained from the same instrument are also good. How-
ever, similarity analysis of the DSC profiles of the
same protein using different instruments has large
SDs. The major sources of poor precision using pro-
file similarity between instruments could be due to
the differences in the melting profile shapes and the
operation environments. Even with proper DSC cali-
brations, only temperature and H (enthalpy change)
are calibrated. The unfolding profile (or peak shape)
is not calibrated. The same peak area may have quite
different profiles. Therefore, it is possible that with
different instruments, some factors such as slight dif-
ferences in sample cell configurations and so on may
affect how the protein unfolds and cause DSC pro-
files to be different. Therefore, in most cases, Tm is
a preferred parameter to determine protein thermal
stability changes.
There is a significant difference in using MicroCal
VP-DSC and MicroCal VP-Capillary DSC (MicroCal
LLC) with regard to the detection of protein precipi-
tation. A MicroCal VP-DSC (MicroCal LLC) typically
shows an exothermic signal after a protein precip-
itates, but a MicroCal VP-Capillary DSC (MicroCal
LLC) does not show such an exothermic signal when
the same protein is studied. In conditions where pro-
tein precipitation are detected using MicroCal VP-
DSC (MicroCal LLC), we typically saw minimal im-
pact for Tm comparison, but more significant impact
for profile similarity comparison. This is consistent
with our observations that precipitation often occurs
around or after the middle point of a thermal transi-
tion; thus, it has less impact on Tm and more impact
on the overall profile.
Replicate Measurements of the Same Protein
Using the Same Instrument on Different Days
The intraday versus the interday precision was also
evaluated. The DSC scans of the intraday and inter-
day measurements of protein 2 samples are shown
in Figure 2, and the results are listed in Table 5. Al-
though the interday precision is slightly larger than
the intraday precision, the differences between the
interday precision and the intraday precision for both
Tm and similarity analysis are still within the SDs of
both parameters using the same instrument (shown
in Tables 2 and 3). This suggests that the DSC instru-
ment is relatively stable over time and the compari-
son study can be performed over several days as long
as the protein is stable. If a longer time is needed,
the instrument should be evaluated accordingly and
a control should be included for the study.
DOI 10.1002/jps
 APPLICATIONS OF DSC FOR THERMAL STABILITY ANALYSIS OF PROTEINS 959

Table 2. Summary of Tm Values and SDs of Replicate Measurements of the Same Protein Using the Same Instrument

Tm (Average from Each Instrument SD) ( C)

Site Protein 1 Protein 2 Protein 3 Protein 4 Protein 5

Site 1 73.22 0.10 56.71 0.10 49.24 0.17 50.80 0.07 46.24 0.01
MicroCal 81.62 0.18 77.77 0.02 68.47 0.10 65.80 0.34
VP-Capillary DSC 83.65 0.10
Site 2 73.27 0.03 56.61 0.03 NAa 52.12 0.12 46.41 0.09
MicroCal 82.62 0.08 77.47 0.01 68.75 0.02 66.79 0.51
VP-DSC 83.61 0.02
Site 3 73.48 0.04 56.25 0.06 49.94 0.59 51.02 0.04 46.97 0.41
MicroCal 82.69 0.14 77.50 0.17 68.62 0.16 65.36 0.13
VP-DSC 83.64 0.16
Site 4 TA 72.30 0.21 57.60 0.68 49.42 1.13 51.87 0.30 44.73 1.07
Q2000 DSC series 82.49 0.94 77.11 0.57 68.35 0.04 66.41 0.14
with RCS coolant 82.30 0.24
Site 1 74.49 0.10 59.94 0.50 50.57 0.36 51.11 0.11 47.28 0.09
MicroCal 82.07 0.29 79.78 0.16 69.33 0.17 66.02 0.25
VP-DSC 84.03 0.09
a The first domain was not detected due to the weak signal.
NA, not applicable.

Replicate Measurements of the Same Protein Sample at
Different Concentrations Using the Same Instrument or
Different Instruments
The protein concentrations used for the TA in-
strument are quite different from the concentra-
tions used for the MicroCal instruments (25 vs.
0.51.0 mg/mL). Even though the five proteins stud-
ied here showed similar Tm values for the TA instru-
ment, wherein proteins were measured at 25 mg/mL,
and the MicroCal instruments (MicroCal LLC),
wherein the same proteins were measured at
0.51.0 mg/mL, the Tm values of a protein at differ-
ent concentrations could still be different depend-
ing on specific proteins. This is due to the fact that
proteins may irreversibly aggregate after thermally
induced unfolding, and this irreversible aggregation
can be protein concentration dependent, affecting the
unfolding and apparent Tm . Therefore, proteins with
different thermodynamic properties may show differ-
ent Tm results when measured at significantly differ-
ent protein concentrations. In addition, some proteins
may show significantly different interactions at differ-
ent concentrations (0.5 vs. 25 mg/mL). In such cases,
the Tm values measured at different concentrations
can also be different.
Qualification of DSC: Sensitivity Assessment
Comparison of Native Proteins with their pH 3 and 6 M
Gdn Denatured Forms
The DSC scans of four selected proteins (protein 6,
IgG1; protein 1, IgG2; protein 7, Fc conjugate; protein
8, helical bundle) in different stable storage buffers

Table 3. Summary of Similarity Values and SDs of Replicate Measurements of the Same Protein Using the Same Instrument

Similarity (Average from Each Instrument SD) (%)

Site Protein 1 Protein 2 Protein 3 Protein 4 Protein 5

Site 1 MicroCal VP-Capillary DSC 99.52 0.07 99.75 0.06 98.73 0.60 99.70 0.07 97.95 0.85
Site 2 MicroCal VP-DSC 99.92 0.02 99.83 0.10 99.77 0.04 99.92 0.02 99.34 0.37
Site 3 MicroCal VP-DSC 96.77 2.31 91.99 2.36 96.39 2.67 99.60 0.12 96.39 1.08
Site 4 TA Q2000 DSC series with RCS coolant 98.37 0.82 97.84 1.00 99.70 0.28 98.58 1.15 99.59 0.55
Site 1 MicroCal VP-DSC 99.04 0.17 95.28 3.76 96.69 2.15 99.52 0.27 97.23 1.38

Table 4. Summary of Tm and Similarity Values of Replicate Measurements of the Same Protein Using Different Instruments

All Sites Protein 1 Protein 2 Protein 3 Protein 4 Protein 5

Tm Values of Each Transition (Average from Each Instrument SD) ( C)
Overall average SD 73.35 0.78 57.42 1.49 49.91 0.67 51.38 0.58 46.33 0.98
82.30 0.45 77.93 1.06 68.70 0.38 65.99 0.60
83.45 0.66
Percentage of similarity values (average from each instrument SD)
All MicroCal DSC instruments Protein 1 Protein 2 Protein 3 Protein 4 Protein 5
Overall average SD 74.4 16.4 44.2 4.8 88.0 8.2 85.8 22.8 88.9 4.4

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960 WEN ET AL.

Table 6. DSC Profile Similarities Compared with the Controls

in the Sensitivity Study

Profile Similarity (%)

Protein Control to Itself C3N to Control Gdn to Control

Protein 1 100 6 0
Protein 6 100 1 6
Protein 7 100 6 8
Protein 8 100 3 3

The experiment was performed on a MicroCal VP-Capillary DSC.

and C3N and 6 M Gdn, wherein the proteins were

unfolded to different extents, are shown in Figures
3a3d, respectively, and the profile similarities com-
pared with the controls are shown in Table 6. For
the proteins in stable storage buffers, there are up to
three apparent endothermic thermal transitions cor-
responding to the unfolding of the individual domains,
demonstrating that the proteins are folded into the
distinct domains under these conditions. In C3N, the
DSC profiles of the proteins change significantly (pro-
file similarity <10%), with different thermal tran-
sition temperatures as compared with those of the
folded proteins, and the enthalpies of these transi-
tions are also significantly lower. These results sug-
gest that the proteins in C3N are partially unfolded
and the transitions are from the unfolding intermedi-
ates, and possible aggregates are formed. In the pres-
ence of 6 M guanidine, there is no thermal transition
observed in the DSC scans of all four selected pro-
teins, suggesting that the domains have unfolded fur-
ther (mostly unfolded as indicated by spectroscopic
analyses, circular dichroism, and Fourier transform
infrared spectroscopy; data not shown), and the pro-
teins have less structure in 6 M guanidine than in
C3N. The DSC results demonstrate that DSC is able
Figure 2. (a) Intraday DSC scans versus (b) interday DSC to detect thermal stability and changes in the domain
scans of protein 2 in formulation buffer. The experiment was conformation in the proteins caused by low pH or 6 M
performed on a MicroCal VP-Capillary DSC. Gdn and is appropriate for assessing protein ther-
mal stability and conformational changes that may
Table 5. Summary of the Intraday Precision Versus Interday result from the manufacturing process as well as for-
Precision of Protein 2
mulation and storage conditions. The above exper-
Intraday Interday iments cannot provide quantitative information to
Tm1 Tm2 Tm1 Tm2
show the sensitivity. Therefore, blending studies of
1 56.59 77.76 56.59 77.76 proteins with different structures in the same buffer
2 56.78 77.79 56.92 77.92 were performed as described in the next section.
3 56.75 77.76 56.66 77.82
Average 56.71 77.77 56.72 77.83 Blending Study of Proteins with Different Structures
SD 0.10 0.02 0.17 0.08 in the Same Buffer
Similarity (%) Similarity (%)
As mentioned before, the challenge for studying the
1 99.81 99.59 sensitivity of DSC by experimentally mixing the na-
2 99.69 98.84 tive and unfolded proteins is that the unfolding of a
3 99.75 98.42
protein by low pH or Gdn is often reversible or par-
Average 99.75 98.95
SD 0.06 0.59 tially reversible. Thus, a standard curve that corre-
lates the changes in the DSC profiles with the ratio
The experiment was performed on a MicroCal VP-Capillary DSC.
of a denatured protein to its native form at various

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 APPLICATIONS OF DSC FOR THERMAL STABILITY ANALYSIS OF PROTEINS 961

Figure 3. DSC scans of four selected proteins(a) protein 6, (b) protein 1, (c) protein 7, and
(d) protein 8in three different buffers: formulation (black), C3N (red), and 6 M Gdn (blue).
The offsets of some scans are adjusted for clarity. The experiment was performed on a MicroCal
VP-Capillary DSC.

mass ratios cannot be generated empirically due to
the changes in the unfolded intermediate (structure
and/or amount) after mixing. To address this issue, we
blended two stable native proteins of different struc-
tures as surrogates for the native and unfolded forms
of the same protein.
The DSC profiles of two proteins (protein 9 and
protein 10) mixed in the same buffer empirically and
mathematically at different mass ratios are shown in
Figure 4. The profile similarity results are presented
in Table 7. The results show that the computer mix-
ing profiles and similarity values agree very well with
the experimental profiles and similarity values ob-
tained empirically, with less than 5% difference at
the same mixing ratios. This suggests that the im-
pact of irreversible reactions is negligible in this case.
This result demonstrates that the computer simula-
tion method may be used for evaluating the sensi-
tivity of DSC. From the results of the protein 9 and
protein 10 mixing study, we can detect the presence
of as low as 10% of denatured protein using the DSC
method. In both cases, the similarity values in Ta-
ble 7 are below 95% (precision of the method). There
are some assumptions and limitations in this method:
The impact of irreversible reactions should be negli-
gible, the proteins should unfold separately and inde-
pendently, and the same buffer should be used for both
proteins.

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962 WEN ET AL.

Figure 5. pH stability of protein 11 in different pH

buffers. The offsets of some scans are adjusted for clarity.
The experiment was performed on a MicroCal VP DSC.

APPLICATIONS
To further demonstrate that DSC is suitable for com-
paring the thermal stability of different proteins in
the same condition and the same protein in different
conditions, several examples are shown below.

Figure 4. Comparison of the computer mixing (a) versus
experimental mixing DSC profiles (b) of protein 9 and pro-
tein 10 samples. Black, 100% protein 9; green, 5% protein
9 + 95% protein 10; cyan, 10% protein 9 + 90% protein 10;
blue, 50% protein 9 + 50% protein 10; dark yellow, 75%
protein 9 + 25% protein 10; red, 100% protein 10. The ex-
periment was performed on a MicroCal VP-Capillary DSC.
pH Stability of Candidates
pH stability is one of the important aspects
that should be evaluated during protein product
development.6 The DSC profiles of protein 11 at dif-
ferent pHs in citrate buffer are shown in Figure 5.
The changes in Tm are greater than the instrument
variability, demonstrating that the DSC method is
able to detect the changes in thermal stability and
overall conformation of the protein. The DSC profiles
in Figure 5 indicate that the order of pH-dependent
thermal stability for this protein is pH 7 pH 6 >
pH 5 > pH 4. A dramatic decrease in stability in cit-
rate buffer occurred at pH 5 and below. The unfolded
protein became very soluble at pH 3 and pH 2.6

Table 7. Comparison of the Profile Similarity of the Computer Mixing Versus Experimental Mixing Samples of the Protein 9
and Protein 10

Mixture Similarity to Protein 10 (%) Similarity to Protein 9 (%)

Protein 10 (%) Protein 9 (%) Computer Experiment Computer Experiment

100 0 100 100 3 3
95 5 97 96 0 1
90 10 89 87 1 3
50 50 29 32 39 35
25 75 9 9 73 67
0 100 3 3 100 100

The experiment was performed on a MicroCal VP-Capillary DSC.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 3, MARCH 2012 DOI 10.1002/jps
 APPLICATIONS OF DSC FOR THERMAL STABILITY ANALYSIS OF PROTEINS
Figure 6. DSC profiles of protein 11 under different buffer
conditions. The offsets of some scans are adjusted for clarity.
The experiment was performed on a MicroCal VP DSC.
Buffer Screening
Differential scanning calorimetry is also a useful tool
for screening the buffers and excipients used during
processing and formulation. The DSC scans of pro-
tein 11 under different conditions (different concen-
trations of NaCl or ammonium sulfate) are shown in
Figure 6. The changes in Tm after adding NaCl or
ammonium sulfate are greater than the instrument
variability, suggesting that the DSC method is able to
detect the changes in thermal stability under differ-
ent solution conditions. The DSC results indicate that
sodium chloride and ammonium sulfate both decrease
the thermal stability and solubility of this protein dra-
matically in acetate buffer, suggesting that exposure
to NaCl and ammonium sulfate should be controlled
during method development, process, and storage.6
Candidate Screening
Many proteins have a propensity to slowly aggregate
and precipitate with time during storage or process-
ing. During the earliest phases of protein therapeutic
development, there are often multiple candidates to
choose from. This provides an opportunity to go for-
ward with the candidate that is most stable to process
and storage conditions. Screening candidates is one of
the ways to ensure that the protein with the greatest
chance of being successfully produced as a therapeu-
tic enters late stage development.
The DSC scans of two candidates (proteins 11 and
12) in different buffers are shown in Figure 7. The
thermal transition temperatures of candidate 1 (pro-
tein 11) are higher than those of candidate 2 (protein
12) in all corresponding buffers, suggesting that the
thermal stability of candidate 1 is greater than that
of candidate 2. In addition, the unfolded intermedi-
DOI 10.1002/jps
963
Figure 7. DSC scans of therapeutic candidate 1 (protein
11; dot lines) and candidate 2 (protein 12; solid lines) in dif-
ferent buffers. Black, 20 mM sodium phosphate, pH 6.5; red,
20 mM sodium phosphate, 0.3 M NaCl, pH 6.5; blue, 20 mM
sodium phosphate, 0.5 M ammonium sulfate, pH 6.5. The
offsets of some scans are adjusted for clarity. The experi-
ment was performed on a MicroCal VP DSC.
ate(s) of candidate 1 are more soluble than those of
candidate 2. The unfolding is an equilibrium reac-
tion, and the aggregation caused by decreased solu-
bility is an irreversible reaction, which may result in
decreased stability. Both thermal stability and solu-
bility data suggest that candidate 1 is a more stable
molecule and has a better chance of remaining folded
during manufacturing, and is, therefore, possibly a
better candidate than candidate 2.6 The DSC results
agree with the results obtained from other techniques
(data not shown).
In summary, DSC has been used to study pro-
teins for formulation, candidate screening, protein
engineering, protein folding, domain stability, pro-
teinprotein and proteinsmall molecule interac-
tions, forced degradation, carbohydrates versus sta-
bility, thermal reversibility, and so on.47,1622 The
work shown above demonstrates that DSC is a precise
and sensitive tool for these applications.
CONCLUSION
The results from the multisite, multi-instrument, and
multi-analyst study demonstrate good precision of
DSC measurements for the Tm values. If the DSC
measurements are from the same instrument, pro-
file similarity values are also high. However, if the
data are from different instruments, the profiles of
the same protein may vary dramatically. The mea-
surements of the Tm values are relatively constant re-
gardless of the site, instrument, or analyst. Replicate
measurements of the same protein using the same
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 3, MARCH 2012
964 WEN ET AL.
instrument on different days show no significant dif-
ferences.
Our sensitivity studies demonstrate that DSC is
able to detect pH- and denaturant-induced changes
in protein thermal stability, and it is appropriate to
use DSC when assessing the thermal stability of a
protein under these denatured conditions. We may be
able to detect as low as 10% denatured protein using
the DSC method.
The applications of DSC in pH screening, buffer
screening, and candidate screening for formulation,
storage conditions, and downstream processing have
further demonstrated that information from DSC
thermal stability analysis can be applied to compara-
bility and characterization studies as well as to eval-
uate conformational stability of proteins.
ACKNOWLEDGMENTS
The authors thank Linda Narhi, Brent Kendrick,
Vladimir Razinkov, and David Brems for their con-
tributions and helpful discussions to this study and
manuscript.
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