Conformational changes of antibodies upon adsorption onto hydrophobic interaction chromatography surfaces

Journal of Chromatography A, 1552 (2018) 60–66

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Conformational changes of antibodies upon adsorption onto
hydrophobic interaction chromatography surfaces
Beate Beyer a,b , Alois Jungbauer a,b,∗
a
b

Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Vienna, Austria
Austrian Centre of Industrial Biotechnology, Vienna, Austria

a r t i c l e

i n f o

Article history:
Received 25 January 2018
Received in revised form 14 March 2018
Accepted 4 April 2018
Available online 5 April 2018
Keywords:
IgG
Immunoglobulin
Differential scanning calorimetry
Isothermal titration calorimetry
Unfolding

a b s t r a c t
Differential scanning calorimetry was established for in-situ measurement of the transition temperatures
of antibodies when adsorbed on hydrophobic interaction chromatography media. This method is also
suitable for non-transparent media, which is an advantage over spectroscopic methods that cannot be
used in many cases due to large background signals. The three transition temperatures of an antibody
were lowered when the molecule was adsorbed onto Phenyl and Butyl functionalized Toyopearl particles
as well as on Phenyl Sepharose 6 Fast Flow when bound at moderate to high salt concentration compared
to the values in free solution. The first two melting points, representing the CH2 domain and the Fab
fragment, are more affected than the highest melting point, which corresponds to the CH3 domain. It
is obvious that domains which are less stable are more likely to undergo conformational change upon
adsorption. It could be shown that the conformational changes occurring in antibodies upon adsorption
to HIC media are directly proportional to the hydrophobicity of the stationary phase and that they are
reversible. Upon elution, the protein returns to its original conformation. For all four tested resins, a
negative value for both H as well as S was calculated, leading to opposing contributions to G.
© 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
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1. Introduction
Hydrophobic interaction chromatography (HIC) is frequently
used because it is an orthogonal method to ion-exchange and
affinity chromatography with a completely different selectively.
One of the most common arguments for using HIC is that this
method is widely regarded as a “non-destructive technique” due
to the absence of organic modifiers and harsh elution conditions [1]. It is commonly believed that proteins can be purified
and analyzed in their native condition by hydrophobic interaction chromatography. Still, this claim has to be taken with a
grain of salt, since over the years many studies have at first suspected and later outright proven, that conformational changes can
occur during the adsorption of proteins onto hydrophobic surfaces [2–6]. Our group proposed a model, according to which a
certain fraction of the injected protein unfolds upon adsorption
onto the HIC stationary phases resulting in stronger binding and
delayed elution from the column [7,8], which would explain the

loss of protein often observed when HIC is used in a purification step [9]. The partial unfolding has been measured in-situ by
Attenuated Total Reflectance Fourier Transformed Infrared (ATR

∗ Corresponding author at: Department of Biotechnology, University of Natural
Resources and Life Science, Vienna, Muthgasse 18, 1190, Vienna, Austria.
E-mail address: (A. Jungbauer).

FT-IR) spectroscopy. Recently, Antos and coworkers [10] suggested
that assuming a reversible unfolding mechanism might be more
accurate for describing the behavior of certain proteins during
adsorption in HIC and supported this claim by Nano Differential
Scanning Fluorimetry measurements.
While these conformational changes upon adsorption have
readily been observed for certain proteins, others do not seem to
be affected. It has been hypothesized that this tendency towards
unfolding upon adsorption is strongly dependent on the type of
the stationary phase used on the one hand and on the structure
and the physical properties of the protein in question on the other
hand. Especially the adiabatic compressibility of the protein has
been suspected to be of decisive influence [8], since it has been
observed that “softer” proteins, which have higher adiabatic compressibility, are more prone towards unfolding and show stronger
retention on hydrophobic surfaces [11].
Most of the available data on this topic has been obtained using
classical model proteins including lysozyme, bovine serum albumin
(BSA), ␤-lactoglobulin, Ca++ depleted lactalbumin or ovalbumin.
This raises the question, if and how molecules that are of higher
interest for the biopharmaceutical sector, such as antibodies, are
affected by the interaction with hydrophobic surfaces that occurs
in HIC. In order to tackle this, we chose a GMP manufactured IgG1
therapeutic antibody as the model protein for this study, which is a

good representative of the majority of molecules in this class of bio-

/>0021-9673/© 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license ( />

B. Beyer, A. Jungbauer / J. Chromatogr. A 1552 (2018) 60–66

therapeutics and compared the adsorption behavior of this protein
towards various commercially available HIC media.
In the past, ATR FT-IR has been used for studying conformational
changes during the adsorption of proteins onto chromatographic
surfaces. This method, however, is only partially suitable for the
purpose, since the measurement is heavily dependent on the optical characteristics of the stationary phase particles. While good
results could be achieved for Sepharose-based materials, polymetacrylate particles could not be analyzed at all due to a lack of
translucency and high background signals [8]. Based on the nature
of calorimetric measurements, it can be expected that DSC measurements are less sensitive to these factors.
As analysis methods for studying the adsorption behavior
of our model antibodies, Isothermal Titration Calorimetry (ITC)
and Differential Scanning Calorimetry (DSC) were chosen. ITC is
a well-established method for quantifying the thermodynamic
parameters associated with the interaction of proteins with all sorts
of ligands and chromatographic stationary phase particles [12–14].
The original method for this was developed by Chen et al. [15–18].
In such an ITC experiment, the heat flow resulting from repeated
injections of protein solution into the measurements cell containing stationary phase slurry is monitored. Based on this heat flow and
on the protein concentration adsorbed per unit stationary phase,
which can be calculated from the adsorption isotherm, it is then
possible to calculate the adsorption enthalpy Hads . As such ITC is
the only method that allows direct measurement of the enthalpy
of adsorption of proteins binding onto chromatographic stationary
phases. As structural changes of a protein generally require energy

in order to occur, a larger extent of conformational changes under
certain conditions should also manifest itself in a change in Hads ,
since the unfolding process requires energy. Especially when conformational changes are involved, enthalpy measurements by ITC
have been shown to yield more consistent results than van’t Hoff
analysis [19].
Differential Scanning Calorimetry on the other hand, allows
a more direct detection of conformational changes. The thermal
unfolding process of proteins occurs over a narrow temperature
window. Thus, it is possible to reliably determine the transition
midpoint between folded and unfolded state, also referred to as the
melting temperature, by measuring the heat absorbed or released
by a sample upon controlled heating in a DSC instrument. This
temperature value is commonly used as a reference number for
protein stability and any differences between the protein in free
solution and the protein in the adsorbed state would strongly indicate conformational changes. For antibodies, usually 2–3 different
transition peaks are observed in DSC experiments, representing the
unfolding events of the different domains of the molecule. The lowest transition temperature can be attributed to the unfolding of the
CH2 domain, the second one corresponds to the antigen-binding
fragment (Fab) and the third one indicates the unfolding of the CH3
domain. For different antibodies, these peaks may however overlap
to varying degrees [20–22].
For this study, the thermal stability of the model antibody was
analyzed in free solution and in the adsorbed state as well as after
adsorption and subsequent elution from a stationary phase by DSC.
Additionally, the thermodynamic quantities G, H and S associated with the binding of the antibody onto the stationary phases
were measured by ITC.

2. Materials and methods
2.1. Materials and chemicals
All chemicals were of analytical grade, unless stated otherwise.

Disodium hydrogen phosphate (1.06580.500) and ammonium sul-

61

fate for buffer preparation (1.0217.500) were purchased from
Merck.
The recombinant monoclonal antibody CH14.18 was kindly provided by APEIRON biologics. It is a mouse-human chimeric IgG1,
produced in Chinese Hamster Ovary (CHO) cells [23].
TOYOPEARL Phenyl–650 M (0019818) and TOYOPEARL
Butyl–650 M (0019802) were purchased from Tosoh Bioscience and
are both based on hydroxylated polymethacrylic polymer beads,
Butyl-S Sepharose 6 Fast Flow (17-0978-10), Phenyl Sepharose 6
Fast Flow (High Sub) (17-0973-10) and Phenyl Sepharose 6 Fast
Flow (Low Sub) (17-0965-10) were obtained from GE Healthcare
Life Sciences.
Decon Labs Decon 90 was purchased from Fisher Scientific
(11761168).
2.2. Differential Scanning Calorimetry (DSC)
For all protein samples, a buffer exchange via ultra-diafiltration
was performed using the corresponding experiment buffer (20 mM
phosphate containing either 0 mM, 400 mM or 800 mM ammonium
sulfate at pH 7.3) as ultra-diafiltration buffer, Amicon Ultra-15
Centrifugal Filter Units with a cut-off of 50 kDa (Merck Millipore
UFC905096) and a Heraeus Multifuge X3 FR centrifuge (Thermo
Scientific).
For the antibody in free solution measurements, the protein was
then diluted to a concentration of 0.25 mg/mL. 650 ␮L of this solution were loaded into the sample cell of a TA- Instruments Nano
DSC instrument (model: 602000), the reference cell was filled with
buffer and a thermoscan from 25 ◦ C to 100 ◦ C with a scan rate of
1 ◦ C/min was performed. The obtained thermogram data was then

analyzed using the TA Instruments NanoAnalyse software and a
two-state scaled fitting model with 3 peaks.
For the measurements of antibody adsorbed onto chromatographic stationary phase particles, a sample volume corresponding
to 180 ␮g of the model antibody in the chosen experiment buffer
was added to 350 ␮L of a 50% slurry of the stationary phase in
the corresponding experiment buffer and further diluted to a total
volume of 700 ␮L. This solution was then incubated under endover-end shaking for 4 h. To remove any non-adsorbed antibody,
the sample was then spinned down in a Fisherbrand HS10022 minicentrifuge, the excess liquid was removed and the particles were
washed 3 times with an equal amount of the corresponding experiment buffer. The suspension of stationary phase particles with
adsorbed protein in experiment buffer was then loaded into the
sample cell of the Nano DSC and measured using the same conditions as for the antibody in free solution.
For the blank measurements with stationary phase particles, but
without protein, a 0.25% slurry of the stationary phase in the corresponding experiment buffer was prepared and loaded into the
Nano-DSC.
In between sample runs, the instrument was cleaned by flushing
with a 1% solution of Decon 90 followed by deionized water.
2.3. Bind-elute experiments combined with DSC analysis
Before buffer exchange to the experiment buffer containing
800 mM ammonium sulfate, a DSC thermogram of the model
antibody in 20 mM phosphate buffer pH 7.3 was measured. Afterwards, antibody and stationary phase both in the experiment buffer
containing 800 mM ammonium sulfate were incubated under endover-end shaking for 4 h. After this incubation half of the sample
was transferred into the sample cell of the calorimeter and a
thermogram of the antibody bound to the stationary phase was
measured. The other half of the sample was centrifuged in an
Eppendorf 5415 R Benchtop Centrifuge, the supernatant liquid was
removed and an equal volume of 20 mM phosphate buffer pH 7.3


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B. Beyer, A. Jungbauer / J. Chromatogr. A 1552 (2018) 60–66

was added to the resin in order to elute the protein from the
particles. This new elution sample was again incubated under endover-end shaking for 1.h. Afterwards, the resin was removed from
the sample by centrifugation and the thermal stability of the eluted
antibody was measured in the calorimeter.

the stationary phase is plotted against the protein concentration
in solution. The resulting curve can be fitted with the Langmuir
adsorption isotherm Eq. (2) in order to be able to calculate values
for q.

2.4. Batch adsorption isotherms

qmax is the maximum concentration of protein that can be adsorbed
to the stationary phase, c is protein concentration in mobile phase
in equilibrium conditions and Ka is the equilibrium binding affinity
constant of the protein.
Gads , the Gibbs energy change associated with adsorption of
protein onto a stationary phase, can be calculated form Eq. (3):

Isotherms were measured in batch adsorption experiments.
Protein and stationary phase were incubated together overnight
in an Eppendorf Thermomixer Comfort at 30 ◦ C and under 750 rpm
constant shaking. Afterwards samples were spinned down in an
Eppendorf 5415 R Benchtop Centrifuge and protein concentration
was measured using an Agilent Cary 60 UV–vis Spectrophotometer.

q = qmax ∗Ka ∗ c/(1 + Ka ∗ c)

(2)

Gads = −R ∗ T ∗ lnKeq

(3)
(8.314 J mol−1

2.5. Isothermal Titration Calorimetry (ITC)
For all protein samples, a buffer exchange via ultra-diafiltration
was performed as already described in Section 2.2
The sample cell of the instrument was filled with a 50% slurry
in the corresponding experiment buffer. The injection syringe was
filled with protein solution with a concentration of either 10 mg/mL
or 15 mg/mL depending on the resin, the ammonium sulfate concentration in the buffer and the therefore expected adsorption
behavior.
At 30 ◦ C, either 20 injections of protein solution with an injection volume of 10 ␮L or 15 injections à 15 ␮L each were performed
with 780 s in between for baseline equilibration. The resulting thermogram was then analyzed using the Origin 6 analysis software for
integration and blank substraction to obtain the corresponding heat
Qads associated with protein adsorption onto the stationary phase.
In between sample runs, the instrument was cleaned by flushing
with a 1% solution of Decon 90 followed by deionized water.

K−1 ), T

the temperaR is the universal gas constant
ture in K and Keq is the equilibrium binding affinity constant. It can
be extrapolated for infinite dilution from Eq. (4), with c being the
protein concentration in solution:
Keq = lim

q

(4)

c→0 c

The entropy Sads can be calculated based on the fundamental
property relation for the Gibbs energy in Eq. (5) with Sads being
the entropy change associated with adsorption of protein onto the
stationary phase.
Gads =

H ads − T S ads

When measuring the heat of adsorption in ITC, it has to be considered, that the raw heat of adsorption of the protein (Q)prot , as
measured by the system, includes contributions from the dilution
heats of the protein (Qdil )prot and the stationary phase (Qdil )sp as
well as the heat of adsorption of the ions present in the buffer
(Qads )ion . These have to be subtracted in order to obtain the net
heat of adsorption Qads as described in Eq. (6).

3. Theory

Qads = (Qads )prot − (Qdil )prot − (Qdil )sp − (Qads )ion

For a classical thermodynamic description of a system three
quantities have to be determined, the Gibbs energy
G, the
enthalpy H and the entropy S.
The adsorption enthalpy Hads can be calculated from the

resulting heat flow in an ITC experiments and batch adsorption
studies based on Eq. (1).

4. Results and discussion

H ads = Q ads /(Vs ∗q)

(1)

Hads is the enthalpy change associated with adsorption of protein
on stationary phases, Qads is the net heat measured in ITC (sum of
area of all injections), Vs is the sorbent volume in the measurement
cell, and q is the protein concentration adsorbed per unit stationary
phase, which can be calculated from the isotherm.
The most frequently used model for fitting adsorption isotherm
data is the Langmuir model. Theoretically the Langmuir model is
not suitable for adsorption data of proteins, if it has to be suspected
that conformational changes might occur upon adsorption. Nevertheless, the model has been used numerous times in the past for
HIC data, achieving acceptable model fits [24–26], also in the context of microcalorimetric experiments [27]. As described in Eq. (1),
what is required for interpreting the results of an ITC experiments
is q, the amount of protein that will bind to the resin at the concentration of protein in the measurement cell after the injections.
If the Langmuir model fits the experimental batch adsorption data
it should be suitable for estimating this value.
For calculating Langmuir adsorption isotherms in batch adsorption studies, the amount of protein bound to the stationary phase
at various total protein concentrations in equilibrium conditions
has to be measured. Then, the concentration of protein bound to

(5)

(6)

4.1. Conformational changes upon adsorption
Conformational change upon adsorption cannot be measured
in situ by ATR FT-IR for certain types of chromatographic media,
such as synthetic polymer based, because they are not transparent. We developed an alternative method based on Differential
Scanning Calorimetry. In order to test how binding onto HIC
chromatographic media affects the conformational stability of antibodies, the thermal stability of a model antibody was measured
first in phosphate buffer both with and without ammonium sulfate
and then adsorbed onto various stationary phases at two different
concentrations of ammonium sulfate. A clear shift of the melting points was observed, depending on the buffer conditions and
whether the molecule was adsorbed to a particle surface or present
in free solution. In free solution in presence of high concentration
of ammonium sulfate (800 mM) a slight increase of the transition temperatures was measured compared to phosphate buffer
with 400 mM ammonium sulfate and phosphate buffer without any
ammonium sulfate. (Fig. 1A and B). This is interpreted that the high
concentration of salts slightly improves the conformational stability. This stabilizing effect of ammonium sulfate is commonly known
and the reason why this salt is widely used as a stabilizing additive
and non-inactivating precipitant for proteins [28,29]. At the lower
concentration of ammonium sulfate no unfolding peaks could be
detected for the sample adsorbed onto Butyl-S Sepharose 6 Fast
Flow, which was probably due to weak binding of the antibody to
this resin under these conditions.


B. Beyer, A. Jungbauer / J. Chromatogr. A 1552 (2018) 60–66

63

Fig. 1. Thermal stability of the model antibody, A: in experiment buffer containing 800 mM ammonium sulfate, B: in experiment buffer containing 400 mM ammonium
sulfate; C: at the different stages of a bind-elute experiment with the TOYOPEARL Butyl–650 M resin at 800 mM ammonium sulfate; TM1, TM2 and TM3 represent the three

transition points of the different domains of the antibody. The error bars represent the standard deviation calculated from three independent measurements of the transition
temperatures.

Fig. 2. DSC raw data graphs A: comparison of the raw data from the model antibody adsorbed onto a stationary phase, in this case TOYOPEARL Butyl–650 M, and in free
solution in the corresponding experiment buffer (20 mM phosphate, 800 mM ammonium sulfate, pH 7.3); B: data from blank experiment with resin only, no antibody
adsorbed; (Exemplarily the blank data for the TOYOPEARL Phenyl resin at 800 mM ammonium sulfate is shown here).

Adsorbed onto the different HIC media on the other hand, the
molecule showed a noticeably lower thermal stability (Fig. 1A and
B). This stability change becomes clearly apparent even from the

raw data of the experiments as a distinct shift in the position of the
unfolding peaks can be observed (Fig. 2). For our antibody only two
peaks are visible in the raw data. Nevertheless, the best model fit


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B. Beyer, A. Jungbauer / J. Chromatogr. A 1552 (2018) 60–66

Fig. 3. Scheme of HIC media used for experiments in this study; Ranked according
to hydrophobicity.

was achieved, when modelling the first peak as two overlapping
transition events, which results in three different TM values even
though only two separate peaks are visible. This is very common for
antibodies since the three transition events occur in close proximity
to each other and may therefore overlap to varying degrees.
For the TOYOPEARL Butyl–650 M resin the corresponding shift
in melting temperatures was most pronounced and a difference

of more than 15 ◦ C compared to the protein in free solution was
observed. This strongly indicates that conformational changes are
induced upon adsorption of the molecule onto the stationary phase
surface, which we and others have hypothesized [5–8,19,30,31].
Among the three transition peaks that were used to model the
experimental unfolding signal, the observed temperature shifts
were larger for transition midpoints 1 (TM1) and 2 (TM2) than for
transition midpoint 3 (TM3) (Fig. 1A and B). This only indicates
that domains, which are less stable in general are also more prone
to change their conformation upon adsorption.
It has been suggested previously, that the extent to which
conformational changes occur upon adsorption of proteins onto
HIC stationary phases strongly depends on the hydrophobicity of
the ligand as well as on the surface coverage of the stationary
phase. While a more hydrophobic type of resin should result in
increased conformational changes, a higher surface coverage or
higher loadings have been observed to correspond to a decrease
in conformational changes of the analyte [32].
With respect to the influence of the stationary phase hydrophobicity, our observations confirm the general assumption that the
conformational changes of the target protein increase with the
hydrophobicity of the ligand. In our data, the model antibody shows
the largest shift in conformational stability when adsorbed onto the
TOYOPEARL Butyl–650 M, which according to the manufacturer,
is more hydrophobic than phenyl-functionalized resins based on
ligand hydrophobicity (Fig. 3). The Butyl-S Sepharose 6 Fast Flow
resin is described by the manufacturer as being the least hydrophobic in the Sepharose 6 Fast Flow series, which also makes it the
least hydrophobic resin tested in this study. No significant difference could be detected between the thermal stability of the
antibody adsorbed onto this resin and the antibody in free solu-

tion at 800 mM ammonium sulfate. At lower concentrations of

ammonium sulfate no unfolding peaks could be detected in the DSC
thermogram for samples adsorbed onto this resin, which was probably due to the very weak binding of the antibody to the stationary
phase under these conditions.
As a result, our observations correspond very well with the
general assumption that more hydrophobic HIC resins induce conformational changes in proteins to a much larger extent.
The striking difference between the results obtained for the two
TOYOPEARL resins as well as the similarity of the values obtained
for all the phenyl functionalized media, make it seem very unlikely
that the backbone of the media has any significant influence. Neither does it seem plausible that any differences in the binding
mechanism of the phenyl and the butyl ligand would contribute
significantly to the observed behavior since the data for the Butyl-S
Sepharose 6 Fast Flow resin correlates very well with the general
trend of lower stationary phase hydrophobicity leading to smaller
shifts in the conformational stability of the adsorbed protein. Also,
the two different concentrations of ammonium sulfate showed little to no effect on the detected conformational stability changes as
long as enough ammonium sulfate was present to facilitate binding.
The second assumption that higher surface coverage of the stationary phase leads to smaller amounts of conformational changes
in the protein raises the question whether different degrees of ligand density on the stationary phase could also play a role in this
phenomenon. Phenyl Sepharose 6 Fast Flow (High Sub) has, according to the manufacturer a higher ligand density than its Low Sub
counterpart. Especially at 800 mM ammonium sulfate the thermal
stability of the model mAb adsorbed onto the Phenyl Sepharose
6 Fast Flow (Low Sub), is slightly higher and therefore closer to
the values obtained in free solution, compared to when the same
molecule is adsorbed onto the Phenyl Sepharose 6 Fast Flow (High
Sub). Even though the difference is noticeable at both concentrations of ammonium sulfate, it is relatively minor compared to the
difference between the butyl and the phenyl functionalized media.
For the other resins in question, there is no detailed information
available from the manufacturer as to the degree of ligand substitution. It is therefore not possible to assess how this factor may
contribute to this data.
When a bind-elute experiment was performed and samples at

all three different stages of the adsorption process (antibody in
free solution before buffer exchange and binding to the stationary phase, antibody adsorbed onto the stationary phase, antibody
again in free solution after elution) were subjected to DSC analysis,
it became apparent that elution of the antibody from the stationary phase surface with phosphate buffer containing no ammonium
sulfate brought the thermal transition temperatures back to their
original values, proofing that the unfolding process happening upon
adsorption is reversible (Fig. 1C).
4.2. Thermodynamics of adsorption
Thermodynamic processes can be characterized by three
parameters: The Gibbs free energy G, the enthalpy H and the
entropy S. ITC is the only method to directly measure the enthalpy
of an adsorption process Hads , since Qads , the heat flow measured
by the instrument is proportional to Hads of the system under
constant pressure conditions as described in Eq. (1). The term q,
which is required for calculation of the enthalpy, can be obtained
from the batch adsorption isotherm, one of which is shown in Fig. 4.
The graphs corresponding to the isotherms of the other resins and a
table with the resulting binding capacities and equilibrium binding
constants can be found in the supplementary material.
Injection of a protein into the measurement cell filled with stationary phase particles allows the measurement of the heat of
adsorption of the protein (Q)prot . Since this term also includes the


B. Beyer, A. Jungbauer / J. Chromatogr. A 1552 (2018) 60–66

65

based the DSC results, which suggest that the contribution of conformational changes to the adsorption enthalpy is minor.
Overall, it has to be stated, that for all the tested resins, a negative
value for both H as well as S was calculated, leading to opposing

contributions to G. This so-called enthalpy-entropy compensation is a phenomenon that has often been observed in complex
biological systems [15].
Since in the described experiments the adsorption of the
antibody to the stationary phase particles seems to occur simultaneously with a change in the conformation of protein, it can be
assumed that the reported values for G and H include contributions from both of these reactions. Any assessment of these
contributions based on the final values is not possible since the limits of the available thermodynamic analysis methods are reached.
5. Conclusion
Fig. 4. Adsorption isotherm at two different concentrations of ammonium sulfate
for Toyopearl 650-M; the isotherm graphs corresponding to the other resins can be
found in the supplementary material.

heat of dilution of the protein (Qdil )prot and the stationary phase
(Qdil )sp as well as the heat flow resulting from the adsorption of
ions present in the buffer (Qads )ion , these contributions have to
be assessed in a series of blank experiments injecting both protein sample into a measurement cell containing only buffer and
buffer into the stationary phase slurry in the absence of protein.
The resulting values then have to be subtracted from the adsorption heat of the protein to calculate the net adsorption heat Qads
according to Eq. (6).
It has been proposed that conformational changes of proteins
occurring upon adsorption to hydrophobic surfaces should clearly
manifest themselves in a shift of Hads towards more positive values, since the unfolding process requires energy to occur [8]. Our
data fits this hypothesis, albeit to a smaller degree than expected.
In the DSC experiment the larges shift in conformational stability and therefore the highest amount of conformational changes
was observed when the model antibody was adsorbed onto the
TOYOPEARL Butyl stationary phase. Indeed, Hads for this resin as
measured in ITC is less negative than for the other tested media,
especially at higher concentrations of ammonium sulfate (Fig. 5).
This indicates that less energy is released as a result of the adsorption. One could therefore speculate that the difference in energy is
consumed by the conformational changes of the protein. However,
the differences in Hads are smaller than what could be expected

DSC was established as a method almost ideally suited for
detecting conformational changes occurring in a protein upon
adsorption onto a chromatographic stationary phase particle. Due
to its good reproducibility, the method is sensitive enough to reliably detect changes to the protein’s conformational stability and
the obtained data shows good agreement with previously observed
trends. The possibility to measure both samples in free solution as
well as protein adsorbed onto stationary phase particles represents
an elegant way to directly compare data from samples at different
stages of the adsorption process, when ATR FT-IR is not an option.
It could be shown that the conformational changes occurring in an
antibody upon adsorption to HIC media are directly proportional to
the hydrophobicity of the stationary phase and that upon elution,
the protein returns to its original conformation.
Comparing adsorption behavior towards different types of stationary phases instead of focusing on one individual resin and
various other influence factors such as temperature and salt
concentration can facilitate finding the optimal setup for a chromatographic process before fine-tuning other running conditions.
Gaining further insight into what happens to a molecule during
the adsorption process to different kinds of surfaces is also of decisive importance for accurately modelling this type of interaction in
order to make predictions about chromatographic behavior.
Acknowledgements
This work has been supported by the Federal Ministry of Science, Research and Economy (BMWFW), the Federal Ministry of

Fig. 5. Energy signatures of adsorption of the antibody onto different HIC stationary phases. Parameters Gads , Hads , T Sads were calculated based on ITC data from
injection of the antibody into the measurement cell filled with different types of HIC media slurry in experiment buffer containing A: 800 mM ammonium sulfate, B: 400 mM
ammonium sulfate.


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B. Beyer, A. Jungbauer / J. Chromatogr. A 1552 (2018) 60–66

Traffic, Innovation and Technology (bmvit), the Styrian Business
Promotion Agency SFG, the Standortagentur Tirol, the Government
of Lower Austria and Business Agency Vienna through the COMETFunding Program managed by the Austrian Research Promotion
Agency FFG.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at />009.
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