Conformational changes of antibodies upon adsorption onto hydrophobic interaction chromatography surfaces

Differential scanning calorimetry was established for in-situ measurement ofthe 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.

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 April 2018 Available online 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 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 (http://creativecommons.org/licenses/by/4.0/) 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: jungbauer@boku.ac.at (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 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- https://doi.org/10.1016/j.chroma.2018.04.009 0021-9673/© 2018 The Authors Published by Elsevier B.V This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) 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 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 Fast Flow (17-0978-10), Phenyl Sepharose Fast Flow (High Sub) (17-0973-10) and Phenyl Sepharose 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 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 ◦ 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 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 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 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 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 62 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 15 ␮L each were performed with 780 s in between for baseline equilibration The resulting thermogram was then analyzed using the Origin 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) 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) 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 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 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 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 64 B Beyer, A Jungbauer / J Chromatogr A 1552 (2018) 60–66 Fig 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 (TM1) and (TM2) than for transition midpoint (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 Fast Flow resin is described by the manufacturer as being the least hydrophobic in the Sepharose 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 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 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 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 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 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 Conclusion Fig 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 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 66 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 https://doi.org/10.1016/j.chroma.2018.04 009 References [1] S Fekete, J.L Veuthey, A Beck, D Guillarme, Hydrophobic interaction chromatography for the characterization of monoclonal antibodies and related products, J Pharm Biomed Anal 130 (2016) 3–18 [2] S.L Wu, A Figueroa, B.L Karger, Protein conformational effects in hydrophobic interaction chromatography Retention characterization and the role of mobile phase additives and stationary phase hydrophobicity, J Chromatogr 371 (1986) 3–27 [3] T Tibbs Jones, E.J Fernandez, ␣-Lactalbumin tertiary structure changes on hydrophob␣ic interaction chromatography surfaces, Journal o-Lactalbumin 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between proteins and hydrophobic solid surfaces in hydrophobic interaction chromatography: effects of salts, hydrophobicity of the sorbent, and structure of the protein,... studies on the interaction mechanism between proteins and hydrophobic solid surfaces in hydrophobic interaction Chromatography: effects of salts, hydrophobicity of the sorbent, and structure of the protein,... 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