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MATEC Web of Conferences 333, 15001 (2021) https://doi.org/10.1051/matecconf/202133315001 APCChE 2019

Isothermal as a Method for Analyzing in Exchange

Joao SIMOES-CARDOSO1, Nanako HOSHINO1, Noriko YOSHIMOTO1, and Shuichi YAMAMOTO1*

1Yamaguchi University, Graduate School of Sciences and for Innovation of Engineering, Faculty of Engineering, 2-16-1 Tokiwadai, Ube-shi, Yamaguchi 755-8611, Japan

Abstract chromatography is a widely used method for purification of all types of biomolecules in current biotechnological downstream processes. Knowledge on the binding behavior of provides valuable insight for understanding the molecular mechanisms of protein interactions in a biological context. However, thermodynamic parameters such as and entropy changes that characterize protein adsorption are still unknown. Isothermal titration calorimetry applications in biosciences has gained its merit to study binding of soluble , protein inhibition, conformational changes, reaction kinetics, and protein adsorption. However, in the case of protein adsorption, a lot of complications arise since the usual models used to study protein interactions in are no longer valid. This explains a detailed methodology for the obtention of adsorption enthalpy, entropy and Gibbs energy of protein adsorption, by using ITC together with equilibrium adsorption isotherms.

1 Introduction existing mechanistic models for adsorption, and stimulate the creation of better downstream purification techniques. Protein therapeutics have increased dramatically in number and frequency of use. Protein therapeutics already have a significant role in almost every field of medicine, 2 Materials and Methods like growth factors, enzymes and , which are being improved by several approaches. Proteins can be 2.1 Materials produced by gene expression methods using different expression systems ranging from bacteria to transgenic The selected model stationary phase was Toyopearl Q- animals. 600C AR (TOSOH Biosciences, Japan) an anion- One of the most challenging factors on the exchanger resin with grafted ligands. Table 1 summarizes biomolecule production is its purification from the physical properties of the model chromatographic culture and most importantly its separation from other surface. Bovine serum albumin Lot#037K0765 was similar proteins that are present in the expression systems. purchased from Sigma-Aldrich (USA). It was noticed that Protein chromatography, a technique based on selective commercially protein was not solely composed by BSA adsorption, is able to successfully purify proteins for in its monomeric isoform but approximately 10% dimer therapeutic use while following the guidelines required by and 90% monomer. Before the use of BSA on isothermal drug regulatory agencies. Downstream protein processing titration calorimetry and equilibrium adsorption isotherms is now routinely found to be the bottleneck in experiments, dimers and monomers were separated using manufacturing because it has not kept a preparative size exclusion chromatography column pace with upstream production latest improvements. In Superdex Hiload 26/600, 200 pn (GE Healthcare, USA). some cases, the lack of capacity All salts and buffers used in this work were purchased can seriously affect the profitability of a new from Wako (Japan) and dissolved in ultrapure deionized pharmaceutical product and even result in its failure. water. Maximum binding capacities and mass transfer

properties have been the main focus point of optimizations by the chromatography industry(Müller 2005). However, knowledge on the binding behavior of proteins, liquid–solid interfaces provides valuable insight

into the molecular mechanisms that will help the development of new chromatography surfaces, expand

* Corresponding author: [email protected] © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). MATEC Web of Conferences 333, 15001 (2021) https://doi.org/10.1051/matecconf/202133315001 APCChE 2019

Table 1. Summary of the physical properties of the resin used It the beginning of the calorimetric titration experiment, in this study. 1 information was provided by the manufacturers. the reference cell was filled with distilled degassed water (Solution B in fig. 1). The main cell was filled with 1.8 Physical properties mL of the slurry resin suspension, i.e., 50% (v/v) Pore size of base matrix [nm]1 75 Particle Size of base matrix [µm]1 100 chromatography resin/buffer (Solution A in fig. 1). The Ion Exchange capacity [eq/L gel]1 0.14 – 0.23 syringe was filled with BSAm solution at 20 mg/mL Ligand attachment chemistry1 Grafted ligands. 3rd (Solution C in fig. 1). The cell was closed and agitated at Generation or 300 RPM at 25 ⁰C After a 5400 seconds’ timeout, for the Type B thermal equilibrium can be reached, ten injections of 10 µL were titrated with the 100 µL syringe from the ITC 2.2 Adsorption isotherm experiments equipment. Injections were carried out with a 600 seconds’ interval from each other. The fluctuations Equilibrium adsorption isotherms were determined with indicated by the instrument were below 0.0002 K. Both the goal of determine more accurately the amount of resin slurries and protein were previously protein adsorbed with each injection, explained later in degassed in a degassing station at 400 mmHg for 10 section 2.3. The resins were washed with ultrapure water minutes (TA Instruments, Delaware, USA). and later with the respective buffer solution by decantation. The resins were used in a 50% resin/buffer suspension (resin slurry). Equilibrium adsorption experiments were carried out in 96-well acrylic plates. First, the wells were filled with 0.3 mL different of BSA monomer (BSAm). Initial protein concentration was measured by at 280 nm and 300 nm in a multi-well plate reader PowerWave XS (Biotek, USA). BSA calibration curves were determined in preliminary experiments using 0.2 and 0.3 volume in each well. Then, 20 µL of resin slurry (50% resin-buffer) was added in each well and stirred inside a temperature controlling chamber for at least 8 hours at 25 ⁰C. Preliminary experiments, in the whole range of tested , showed that equilibrium was reached after Figure 1. Schematic representation of a typical isothermal only 15 minutes (data not shown). After equilibration, the titration calorimetry. Solution A: 50% (v/v) resin/buffer. contents of the wells were transferred to a 96-well filter Solution B: pure water. Solution C: BSAm 20 mg/mL. plate and centrifuged for 3 minutes at 2000 RPM onto a new acrylic 96-well plate. 0.2 mL of the filtered solution was transferred to analogous wells in order to keep the 3 Results and Discussion solution pathlength constant in every well. The equilibrium concentration was determined using the same 3.1 Isothermal titration calorimetry method as the initial concentrations. From the equilibrium concentration and initial concentration, the surface The enthalpy change of a system is defined as the sum of concentration could be determined by a mass balance. the change in of that system ( ) and the Equilibrium concentration was plotted against the surface change in the total volume ( ): concentration calculated. The experiments were done in ∆𝑈𝑈 duplicate. = ∆𝑉𝑉+ (1)

2.3 Isothermal titration calorimetry Protein adsorption∆𝐻𝐻 ∆ 𝑈𝑈 occurs∆𝑉𝑉 in virtually incompressible aqueous media ( = 0), this renders Microcalorimeter used in this work was Nano ITC from essencially synonymous with the change in internal TA Instruments (Delaware, USA). It has been already energy ( = ). By definition:∆ 𝑉𝑉 ∆𝐻𝐻 shown that even small pH differences between the ITC cell and titration solution affects quite significantly the ∆𝐻𝐻 ∆𝑈𝑈 = (2) calorimetric results as artifacts in the enthalpy can arise due to buffer protonation effects. Prior to the experiment, where is the ∆ 𝑈𝑈exchange∆𝑄𝑄 − of∆ 𝑊𝑊the adsorption reaction BSAm solutions were prepared in the respective buffers and is the change in work which is approximately and exhaustively washed with the respective buffer using zero in∆ molecular𝑄𝑄 reactions ( 0). Leading to AmiconR Ultra-4 10000MW (Milipore Sigma, USA), for ∆𝑊𝑊 15 min at 7000 RPM. This step was repeated until the =∆ 𝑊𝑊 ≈ (3) conductivity of the buffer solution and protein solution was the same (around 4 to 5 times). Conductivity was ITC directly measure∆𝐻𝐻 s ∆𝑄𝑄 , so it can effectively checked after every washing step with a portable measure the change of enthalpy of protein adsorption. conductivity meter B-173 Twin Cond acquired from ∆𝑄𝑄 Horiba (Japan).

2 MATEC Web of Conferences 333, 15001 (2021) https://doi.org/10.1051/matecconf/202133315001 APCChE 2019

3.2 Infinite dilution and reference state injected solution(Duff, Grubbs, and Howell 2011). Additional contributions to the calorimetric signal can The following well known fundamental thermodynamic arise from buffer interactions that are independent of the relation is used in the subsequent derivations already adsorption interactions. These contributions that reflect derived by previous authors (Atkins and De Paula 2010; temperature dependence of buffer ionization as well as of Blaschke et al. 2011): water structure, need to be subtracted from the total measured signal. Considerable attention must be given to the design of adequate control experiments to clarify these = = (4) contributions(Winzor and Jackson 2006a). The 𝜇𝜇 𝐺𝐺 𝜕𝜕 � � 𝜕𝜕 � � contribution of heat of a protein injection into a resin 𝑇𝑇 , 𝑇𝑇 , 𝐻𝐻 � � � � − 2 mixture using ITC can be divided into several origins: 𝜕𝜕𝜕𝜕 𝜕𝜕𝜕𝜕 𝑇𝑇 𝑝𝑝 𝑛𝑛 𝑝𝑝 𝑛𝑛 where the subscripts and refer to constant = + + and constant composition (closed system). The chemical (11) potential in aqueous solutions𝑝𝑝 𝑛𝑛 ( ) can be described by 𝑒𝑒𝑒𝑒𝑒𝑒 𝑎𝑎𝑎𝑎𝑎𝑎 𝑑𝑑𝑑𝑑𝑑𝑑 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 ∆exp𝐻𝐻 ∆𝐻𝐻 ∆𝐻𝐻 ∆𝐻𝐻 where ∆H is the total enthalpy measured by the ITC. ∆H0 is the enthalpy contribution from the adsorption of = + ln𝜇𝜇 ( ) (5) the protein into resin. ∆Hdil is the contribution of the 0 𝑖𝑖 𝑖𝑖 protein solution dilution heat, measured by titrating For infinte dilution𝜇𝜇 𝜇𝜇 ( 𝑅𝑅=𝑅𝑅 1),𝐶𝐶 this𝛾𝛾 equation formally protein solution into the same buffer that it was diluted reduces to 𝑖𝑖 into. ∆Hsolv is caused by the adsorption of buffer and 𝛾𝛾 it was determined titrating buffer into the resin slurry. = + ln (6) Figure 1 represents typical thermograms obtained for the ∞ 0 ∞ 𝑖𝑖 main experiment and blank trials. So, the blank heat trial where 𝜇𝜇 𝜇𝜇 𝑅𝑅𝑅𝑅 𝐶𝐶 were subtracted from the one obtained titrating the protein

into the resins, and effective enthalpic contribution from = lim (7) the protein adsorption was obtained. ∞ 𝑖𝑖 𝑖𝑖 The first peak of every trial is not shown and it was 𝐶𝐶 𝐶𝐶𝑖𝑖→0 𝐶𝐶 dividing eq. 6 by T results in not used in any calculation due to the well-known phenomenon, named “first-peak anomaly” in previous studies (Werner et al. 2012). Protein injections and all = + ln (8) ∞ 0 blank trials were done in duplicate. 𝜇𝜇 𝜇𝜇 ∞ Adsorption were measured directly by ITC, and 𝑅𝑅 𝐶𝐶𝑖𝑖 and differentiating𝑇𝑇 with 𝑇𝑇respect to the respect to the molar enthalpy of adsorption, ∆hads, was then calculated temperature T under consideration of equation 4 it follows by the following equation:

Δhads=ΔHads/Δn (12) = (9) ∞ 0 𝐻𝐻 𝐻𝐻 where ∆n is the amount of adsorbed upon each − 2 − 2 and thus, 𝑇𝑇 𝑇𝑇 injection (mol), which was calculated using a combination of the equilibrium adsorption isotherm and a mass balance. = (10) ∞ 0 Hence, if the enthalpy𝐻𝐻 obtained𝐻𝐻 calorimetrically was obtained in infinite or close to infinite dilution equals the enthalpy in the reference state

= = (11) ∞ 0 𝑎𝑎𝑎𝑎𝑎𝑎 In practice,∆ 𝐻𝐻this means∆𝐻𝐻 that∆𝐻𝐻 in order to use and compare thermodynamic parameters with enthalpy obtained using van’t Hoff analysis we should determine the adsorption enthalpy at low concentrations ( 0, 0) or take data points at several concentrations and 𝑠𝑠 extrapolate the observed to infinite dilution (Werner,𝐶𝐶 → 𝑙𝑙 𝐶𝐶Blaschke,→ and Hasse 2012;0 Werner, Hackemann, and Figure 1. Thermograms obtained titrating BSA monomer at 20 Hasse 2014). ∆𝐻𝐻 mg/mL onto Toyopearl Q600AR and the respective blank trials. During the calorimetric measurements, a small Buffer was 10 mM TRIS, 30 mM NaCl at pH 7, 25⁰C. signal is observed upon injecting the same solution onto itself. This signal was previously considered to be due to friction, dissipation and other effects like a small mismatch of temperature of the cell and that of the

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3.3 Calculation of the amount of molecule where is the protein concentration in the injection adsorbed upon each injection in ITC syringe (mg/mL); is the injection volume of each 0 injection;𝐶𝐶 and and are the volume of liquid and resin 0 inside the ITC cell𝑉𝑉 (mL), respectively. But, with each 3.3.1 Equilibrium adsorption isotherm fitting 𝑙𝑙 𝑠𝑠 injection ( ), the𝑉𝑉 total 𝑉𝑉liquid volume increases by . Also, after the first injection, one should take into consideration Even a polynomial equation with no physical meaning can 0 be used to calculate the amount of molecule adsorbed the amount𝑖𝑖 of protein that was previously adsorbed.𝑉𝑉 Thus, upon each injection in ITC (Blaschke et al. 2011; the following mass balance equation can be obtained for Hackemann and Hasse 2017). In this work, the well- any given injection: known Langmuir equation was used to relate the equilibrium liquid concentration with surface , + [ + ( 1) ] + (15) concentration upon adsorption: = . ( + ) + . 𝑠𝑠 𝑖𝑖−1 𝑠𝑠 𝑖𝑖−1 𝑙𝑙 0 0 0, , 𝐶𝐶 𝑉𝑉 𝐶𝐶 𝑉𝑉 𝑖𝑖 − 𝑉𝑉 𝐶𝐶 𝑉𝑉 𝑖𝑖 𝑙𝑙 0 𝑠𝑠 𝑖𝑖 𝑠𝑠 𝑖𝑖 . . Since , 𝐶𝐶 𝑉𝑉can 𝑉𝑉be neglected𝐶𝐶 𝑉𝑉 in the = (13) 1𝑚𝑚+ . bracketed terms of the equation. Calculations were made 𝑞𝑞 𝐾𝐾 𝐶𝐶 𝑉𝑉𝑜𝑜 ≫ 𝑉𝑉𝑙𝑙 𝑉𝑉0 𝐶𝐶𝑠𝑠 without neglecting V0, and there was no difference where is the surface concentration𝐾𝐾 𝐶𝐶 (mg of protein/mL between using these terms or not (data not shown). The of resin); is the maximum capacity (mg of protein/mL following system of two equations and two variables can 𝑠𝑠 of resin);𝐶𝐶 K is distribution coefficient; and C is liquid be used from = 0 to = 10. Solving this system for all 𝑚𝑚 concentration𝑞𝑞 after the equilibrium has been reached injections we have solid and liquid phase concentrations (mg/mL). It is important to note that Langmuir equation in every injection.𝑖𝑖 𝑖𝑖 was not developed for protein adsorption studies and assumes several facts that hold no truth in the case of , + + = . + , . biomolecule adsorption onto solid phases. Langmuir . . 𝑠𝑠 𝑖𝑖−1 𝑠𝑠 𝑖𝑖−1 𝑙𝑙 0 0 𝑖𝑖 𝑙𝑙 𝑠𝑠 𝑖𝑖 𝑠𝑠 (16) himself wrote “Considering the nature of the simplifying 𝐶𝐶 𝑉𝑉 𝐶𝐶 𝑉𝑉, = 𝐶𝐶 𝑉𝑉 𝐶𝐶 𝑉𝑉 𝐶𝐶 𝑉𝑉 1𝑚𝑚+ . 𝑖𝑖 assumptions made in its derivation it should, of course, � 𝑠𝑠 𝑖𝑖 𝑞𝑞 𝐾𝐾 𝐶𝐶 𝐶𝐶 𝑖𝑖 not be looked upon as a general equation of the adsorption Lastly, the number of 𝐾𝐾molecule𝐶𝐶 s adsorbed upon isotherm” (Rabe, Verdes, and Seeger 2011). Figure 2 each injection (mol), , can simply be obtained from the represents a typical Langmuir equation fitting. difference of surface concentration obtained in consequent injections:∆ 𝑛𝑛

( , , ) = (17) 𝑠𝑠 𝑠𝑠 𝑖𝑖 𝑠𝑠 𝑖𝑖−1 𝑉𝑉 𝐶𝐶 − 𝐶𝐶 ∆𝑛𝑛 𝑟𝑟 where is the molecular weight𝑀𝑀 of the protein of study (mg/mol). 𝑟𝑟 𝑀𝑀

ads 3.3.3 Dependency between Δh and Cs Figure 3 shows the resulting Δhads from each injection and the surface concentration at which were obtained. As explained in section 3.2, the first peak is ignored due to observable differences. We believe this happens due to Figure 2. Equilibrium adsorption isotherm obtained at 25 ⁰C. two reasons: (1) diffusion of protein from the syringe in BSA monomer at several concentrations were adsorbed onto the equilibration step, and (2) possible air micro bubbles Toyopearl Q600AR, 8 hours later the equilibrium liquid phase contained in the end of the syringe that are released in first concentration was determined. Buffer was 10 mM TRIS, 30 injection. mM NaCl at pH 7, 25⁰C. Resulting fitting parameters: qm = 167.2, K=72.2. As explained in section 3.2, in order to calculate following thermodynamic parameters by the generally know Gibbs-Helmholtz equation: 3.3.2 Mass balance = (18) Since ITC cell and the syringe is a closed system, we may 𝑎𝑎𝑎𝑎𝑎𝑎 𝑎𝑎𝑎𝑎𝑎𝑎 𝑎𝑎𝑎𝑎𝑎𝑎 assume preservation of mass in order to calculate solid where , ∆𝐺𝐺 and∆ 𝐻𝐻T represent− 𝑇𝑇∆𝑆𝑆 the change on the phase concentration with each injection. In the case of the standard free𝑎𝑎𝑎𝑎𝑎𝑎 Gibbs𝑎𝑎𝑎𝑎𝑎𝑎 energy upon adsorption, the change first injection, a simple mass balance equation can be on the ∆standard𝐺𝐺 ∆𝑆𝑆 entropy and the absolute temperature, obtained: respectively. In order to use this relation, that assumes

infinite dilution conditions, it is necessary to extrapolate = . + . (14) the to the intersect ( 0). Figure 3 represent

𝐶𝐶0𝑉𝑉0 𝐶𝐶 𝑉𝑉𝑙𝑙 𝐶𝐶𝑠𝑠 𝑉𝑉𝑠𝑠 𝑎𝑎𝑎𝑎𝑎𝑎 ∆𝐻𝐻 𝐶𝐶𝑠𝑠 →

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two slightly different examples. At pH 8.5, no extrapolation is needed because Δhads is constant = + ln ( ) throughout the experiment (confirming infinite dilution (21) 0 𝑆𝑆 𝑠𝑠 conditions). At pH 7, the effective enthalpy was assumed 𝐶𝐶 𝛾𝛾 ∆𝐺𝐺 ∆𝐺𝐺 𝑅𝑅𝑅𝑅 𝑙𝑙 𝑙𝑙 to be the one determined with injection 2 and 3, while we In practice the ratio of activity coefficients𝐶𝐶 𝛾𝛾 may be taken can assume infinite dilution conditions. as unity ( / 1) if we assume infinite dilution conditions to approach thermodynamic ideality. In 𝑠𝑠 𝑙𝑙 equilibrium,𝛾𝛾 𝛾𝛾 =≈0 in equation 21, the standard free energy change under conditions of thermodynamic ideality is related∆𝐺𝐺 to the equilibrium concentrations of participating species by the expression

= ln ( 𝑒𝑒) (21) 0 𝑆𝑆 𝐶𝐶 𝑒𝑒 ∆𝐺𝐺 −𝑅𝑅𝑅𝑅 𝑙𝑙 where the superscripts are𝐶𝐶 used to identify concentrations at adsorption . Since the distribution coefficient K𝑒𝑒 is by definition the ratio of equilibrium concentrations, equation 21 can be expressed as

= ln (22) Figure 3. BSA adsorption onto toyopearl Q600C-AR. 𝑎𝑎𝑎𝑎𝑎𝑎 obtained from isothermal titration calorimetry at each injection.𝑎𝑎𝑎𝑎𝑎𝑎 ∆𝐺𝐺 −𝑅𝑅𝑅𝑅 𝐾𝐾 Buffer was 10 mM TRIS, 30 mM NaCl () pH 7 and (∆ℎ) pH On the other hand, if described with the dissociation ratio 8.5. = / , the relationship becomes 𝑒𝑒 𝑒𝑒 𝑑𝑑 𝑙𝑙 𝑠𝑠 𝐾𝐾 𝐶𝐶 𝐶𝐶 = ln (23) 𝑎𝑎𝑎𝑎𝑎𝑎 𝑑𝑑 3.3.4. Gibbs energy determination Distribution ∆coefficient𝐺𝐺 𝑅𝑅𝑅𝑅 K 𝐾𝐾can be determined For a reaction to occur spontaneously, like adsorption, the experimentally via several methods, such as, isocratic Gibbs free energy of the initial state ( ) is mode, application of GH-IR model to linear necessarily higher than that of the state towards gradient elution mode(Yamamoto 2005), and as an 𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 example stated in this short study, from equilibrium which the reaction is heading. In an equation 𝐺𝐺form: 𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓 adsorption isotherms fitting. Consequently, of 𝐺𝐺 BSAm adsorption onto Toyopearl Q600C-AR𝑎𝑎𝑎𝑎𝑎𝑎 was = 0 (19) determined and shown in table 2. ∆𝐺𝐺

𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓 𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 where is ∆𝐺𝐺the 𝐺𝐺amount− 𝐺𝐺of energy≤ available for chemically driven work. The change in Gibbs free energy 3.3.5. Determination of the change on adsorption differs from∆𝐺𝐺 the enthalpy change ( ) in that it does not entropy include contributions to energy arising from volume or pressure changes, nor does it ∆include𝐻𝐻 the entropic Although is enough to define the energetics of contributions ( ). protein adsorption,𝑎𝑎𝑎𝑎𝑎𝑎 there are considerable advantages in ∆𝐺𝐺 In liquid chromatography of biomolecules, its breakdown into enthalpic ( ) and entropic temperature, pressure𝑇𝑇∆𝑆𝑆 and of the contributions ( ) that can be represented𝑎𝑎𝑎𝑎𝑎𝑎 by equation ∆𝐻𝐻 usually remain constant. Under these conditions, 18. 𝑎𝑎𝑎𝑎𝑎𝑎 Gibbs energy of the adsorbing protein ( ) is related to its The behavior∆𝑆𝑆 of proteins in adsorption is a net result molar concentration ( ) and protein activity coefficient of various types of interactions already mentioned in 𝑖𝑖 ( ) by the following equation(Winzor𝐺𝐺 and Jackson previous section. If the entropy term contributes to in 𝑖𝑖 2006b): 𝐶𝐶 such a way that it becomes negative, the process is called 𝑖𝑖 ∆𝐺𝐺 𝛾𝛾 entropy driven; if exothermic enthalpy values are = + ln ( ) dominant, leading to a negative , the process is (20) 0 0 considered enthalpically driven. 𝑖𝑖 𝑖𝑖 𝑖𝑖 𝑖𝑖 ∆𝐺𝐺 where is the∆ 𝐺𝐺standard∆𝐺𝐺 free𝑅𝑅 𝑅𝑅energy𝐶𝐶 𝛾𝛾defined under the From enthalpic contributions, we can gain molecular insight on the nature of the predominant constrains0 of constant temperature and solvent chemical 𝑖𝑖 noncovalent interactions responsible for adsorption. For potential𝐺𝐺 (reference state); R is the gas constant and T is example, hydrogen bonding and ionic interactions are the absolute temperature. A simple protein adsorption reaction can be written generally enthalpically driven (negative ) whereas as where and is the free protein in liquid phase hydrophobic interaction derive their strength𝑎𝑎𝑎𝑎𝑎𝑎 from a ∆𝐻𝐻 and solid phase, respectively. The free energy change, positive that outweighs the consequences of any 𝐿𝐿=⇆ 𝑆𝑆 can𝐿𝐿 be written𝑆𝑆 as: enthalpic contributions.𝑎𝑎𝑎𝑎𝑎𝑎 ∆𝑆𝑆 ∆𝐺𝐺 𝐺𝐺𝑆𝑆 − 𝐺𝐺𝐿𝐿

5 MATEC Web of Conferences 333, 15001 (2021) https://doi.org/10.1051/matecconf/202133315001 APCChE 2019

Currently there is no list or detailed separation Müller, E.; “Properties and Characterization of High between enthalpically driven events from entropically Capacity Resins for Biochromatography,” Chemical driven ones due to their inseparable nature. The enthalpy Engineering & Technology, 28(11), 1295–1305 (2015) change depends on the entropy change and vice versa. All things considered, can be directly Rabe, M., D. Verdes, and S. Seeger; “Understanding obtained using equation (18) if 𝑎𝑎𝑎𝑎𝑎𝑎 and were Protein Adsorption Phenomena at Solid Surfaces,” obtained in the same experimental𝑇𝑇∆𝑆𝑆 conditions𝑎𝑎𝑎𝑎𝑎𝑎 assuming𝑎𝑎𝑎𝑎𝑎𝑎 Advances in Colloid and Interface Science, 162(1–2), 87– infinite dilution. Resulting in ∆the𝐻𝐻 calculation∆𝐺𝐺 of the 106 (2011) following thermodynamic parameters shown in table 2. Werner, A., T. Blaschke, and H. Hasse; Table 2. Thermodynamic parameters for BSAm adsorption “Microcalorimetric Study of the Adsorption of PEGylated onto Toyopearl Q600C-AR determined with ITC together with Lysozyme and PEG on a Mildly Hydrophobic Resin: equilibrium adsorption isotherms. 30 mM TRIS, 30 mM NaCl Influence of Ammonium Sulfate,” Langmuir, 28(31), pH 7, at 25 ⁰C. 11376–11383 (2012)

kJ/mol -178.6 Werner, A., E. Hackemann, and H. Hasse; “Temperature K𝒂𝒂𝒂𝒂𝒂𝒂 mL liquid phase / mL resin 72.2 Dependence of Adsorption of PEGylated Lysozyme and ∆𝑯𝑯 kJ/mol -10.6 Pure Polyethylene Glycol on a Hydrophobic Resin: 𝒂𝒂𝒂𝒂𝒂𝒂 kJ/mol/T 0.56 Comparison of Isothermal Titration Calorimetry and van’t ∆𝑮𝑮𝒂𝒂𝒂𝒂𝒂𝒂 Hoff Data,” Journal of Chromatography A, 1356, 188– ∆𝑺𝑺 196 (2014) 4 Conclusion The adsorption enthalpy of the binding/partition process Winzor, D. J. and C. M. Jackson; “Interpretation of the of proteins with ionic exchange are measured by ITC and Temperature Dependence of Equilibrium and Rate reported in this study. K can be calculated via several Constants,” JMR, 19, 389–407 (2006) methods, in this case it was calculated by a Langmuir equation fited to the equilibrium binding isotherm. Yamamoto, S.; “Electrostatic Interaction One of the drawbacks of ITC is that the adsorption Chromatography Process for Protein Separations: Impact process is divided at least five sequential processes, of Engineering Analysis of Biorecognition Mechanism on however, ITC has only capacity of showing the overall Process Optimization,” Chemical Engineering and heat of adsorption and cannot discern different Technology, 28(11), 1387–1393 (2011) subprocesses of adsorption in a chronological manner. This means that the researcher should be careful not to conclude that the major contributions are the only contributions for adsorption, i.e., just because is negative does not mean that entropy is irrelevant for 𝑎𝑎𝑎𝑎𝑎𝑎adsorption. ∆𝐻𝐻 The interaction mechanism of proteins with ion exchange chromatography resins can be studied with ITC and this technique is advantageous in the study of protein adsorption.

5 References Atkins, P. W. and Julio De Paula; 2010, Atkins’ Physical , Oxford University Press (2019)

Blaschke, T., J. Varon, A. Werner, and H. Hasse; “Microcalorimetric Study of the Adsorption of PEGylated Lysozyme on a Strong Cation Exchange Resin,” Journal of Chromatography A, 1218(29), 4720–26 (2019)

Duff, M. R., J. Grubbs, and E. E. Howell; “Isothermal Titration Calorimetry for Measuring Macromolecule- Ligand Affinity,” J. Vis. Exp., 55, https://doi.org/10.3791/2796 (2011)

Hackemann, E. and H. Hasse; “Influence of Mixed Electrolytes and PH on Adsorption of Bovine Serum Albumin in Hydrophobic Interaction Chromatography,” Journal of Chromatography A, 1521, 73–79 (2017)

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