Noninvasive method for determination of immobilized protein A

The pH transition method, developed for the determination of the ion-exchange group density on chromatographic stationary phase, was used for the quantification of immobilized protein A. Monolithic epoxy polyHIPE and particulate CNBr-Sepharose supports were used for immobilization.

Journal of Chromatography A 1671 (2022) 462976 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Short communication Noninvasive method for determination of immobilized protein A Rok Mravljak a, Metka Stanticˇ a, Ožbej Bizjak a, Aleš Podgornik a,b,∗ a Department of Chemical Engineering and Technical Safety, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana SI-1000, Slovenia b COBIK, Mirce 21, 5270 Ajdovšˇcina, Slovenia a r t i c l e i n f o Article history: Received December 2021 Revised 12 March 2022 Accepted 14 March 2022 Available online 15 March 2022 Keywords: Direct noninvasive immobilized protein quantification Protein A pH transition method polyHIPE CNBr-Sepharose a b s t r a c t The pH transition method, developed for the determination of the ion-exchange group density on chromatographic stationary phase, was used for the quantification of immobilized protein A Monolithic epoxy polyHIPE and particulate CNBr-Sepharose supports were used for immobilization A lactate buffer was selected, having a buffer capacity peak approximately 0.5 pH units below the maximum buffer capacity of protein A The pH transition measurements were performed at pH 4.3, where protein A exhibits maximum buffer capacity, with a lactate buffer concentration of mM for protein A immobilized on polyHIPE monoliths and of mM for protein A immobilized on CNBr-Sepharose The pH transition height and full width at half maximum for the particulate support and the height for the polyHIPE matrix, showed a linear correlation with the amount of immobilized protein A determined from the absorbance difference before and after immobilization for both supports The developed method allows a simple, non-invasive online determination of immobilized protein A using biological buffers, even for chromatographic columns with an amount of immobilized protein A as low as 0.25 mg In addition, its sensitivity and duration can be easily adjusted by varying the buffer concentration and pH © 2022 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Introduction Protein immobilization is important in many different fields where specific detection of different molecules or their conversion is required [1] Therefore, they are used either as enzyme bioreactors for conversion of substrates or as ligands for target molecule adsorption [2,3] A plethora of different protein affinity ligands are immobilized on small volume devices such as enzyme-linked immunosorbent assays (ELISA) [4], microfluidic devices [5], and sensors [6], while their high cost dictates that very few are implemented on large-scale processes, among which protein A significantly outperforms all others [7] Various protein A molecules [8– 11] are routinely used for chromatographic purification of monoclonal antibodies on different types of chromatographic supports [9,12–18], thus optimization of the immobilization procedure leading to a maximum ligand utilization is of utmost importance [19– 21] In addition, non-invasive characterization of chromatographic protein A affinity columns is preferred because they are frequently used in a good manufacturing practice (GMP) environment [22] ∗ Corresponding author at: Faculty for Chemistry and Chemical Technology, University of Ljubljana, Vecˇ na pot 113, 10 0 Ljubljana, Slovenia E-mail address: ales.podgornik@fkkt.uni-lj.si (A Podgornik) The methods used for quantification of immobilized proteins are destructive or non-destructive and measure the immobilized protein directly or indirectly Elemental analysis [5], protein hydrolysis in acid at elevated temperature [23], and time-of-flight secondary ion mass spectrometry [24] are examples of direct destructive methods and are therefore limited to a particulate type of stationary phase On the other hand, direct non-destructive methods such as dye binding [25], inhibitor radiolabeling [26], and concentration determination using a bio-assisted potentiometric multisensory system [27] are difficult to implement on largescale or may contaminate the matrix [28] To avoid these limitations, indirect methods are often used to determine the remaining non-immobilized protein, such as the Biuret [29], Lowry [30,31], Bradford (CBB) [32], and Smith (BCA) [33] methods or UVVis absorbance at 280 nm However, these methods might in some cases provide too high estimated value of immobilized protein, when part of immobilizing protein is non-specifically adsorbed and might elute with change of mobile phase Furthermore, they might also be biased by agglomeration of immobilizing protein The amount of immobilized protein can be directly assessed by determining its biological activity, which, however, is not necessarily directly proportional to the quantity of immobilized protein because it exhibits a non-linear dependence at high ligand den- https://doi.org/10.1016/j.chroma.2022.462976 0021-9673/© 2022 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) R Mravljak, M Stanticˇ , O Bizjak et al Journal of Chromatography A 1671 (2022) 462976 sity [34] and also depends on the immobilization procedure, which affects ligand utilization [35] For this reason, direct, noninvasive methods to determine the amount of immobilized protein A would be beneficial Recently, a pH-based method originally developed for the quantification of ion-exchange groups on a matrix has been extended to other groups with ionizable moieties [36] The method is based on the pH transition formed due to the buffering capacity of the ionizable functionalities present on the matrix when the mobile phases with the same pH but different ion concentrations are exchanged stepwise [36–40] In this work, we investigated whether this method can be used for the quantification of immobilized protein A by exploiting its ionizable character Using lactate buffer at pH 4.3, it was possible to determine the amount of immobilized protein A, even on small volume columns comprised of a particulate or monolithic matrix 2.3 Protein A immobilization on CNBr-Sepharose particles Protein A was immobilized on CNBr–activated SepharoseTM Fast Flow particles according to the manufacturer’s instructions [43] Initially, 250 mg of particles were weighed into each of three 15 ml centrifugation tubes All buffers and solutions were filtered and conditioned to 4°C before use The particles were washed three times with ml of cold mM HCl and centrifugation at 1500 G for (Tehtnica Centric 350, Železniki, Slovenia) was used to settle the particles in between washes Subsequently, the particles were washed with the immobilization buffer (0.1 M Na2 CO3 /HCl, 0.5 M NaCl, pH 7.4) using the same procedure After buffer removal, 0, 1, and ml of protein A solution with a concentration of mg/ml were added to the centrifugation tubes, followed by 5, 4, and ml of the immobilization buffer, respectively, and mixed carefully with a pipette Initial absorbance was measured at 280 nm, as a higher concentration of protein A was used compared to polyHIPE immobilization Immobilization was continued until a constant absorbance at 280 nm was reached The difference was used to calculate the amount of immobilized protein A, again considering the initial dilution due to the buffer present in the particle pores The particles were then washed again three times with the immobilization buffer, centrifuged each time in-between To block the remaining CNBr groups, ml of 0.1 M Tris/HCl at pH was added under gentle mixing and allowed to stand at room temperature for hours The particles were then washed alternately three times with an acidic (0.1 M acetate/HCl, 0.5 M NaCl at pH 4) and a basic (0.1 M Tris/HCl, 0.5 M NaCl at pH 8) buffer and stored at 4°C in storage buffer (20 mM phosphate/HCl, pH 7.4) before use For the pH transition experiments, the particles were packed into glass columns using a storage buffer Packing uniformity was verified with a pulse response experiment using 0.5 vol.% acetone as a tracer and a 30 μl loop using HPLC system (ÄKTAExplorer, GE Healthcare, Uppsala, Sweden) at a flow rate of ml/min Materials and methods 2.1 Chemicals Acetic acid, lactic acid, tris(hydroxymethyl)aminomethane (Tris) were purchased from Sigma-Aldrich (St Louis, USA) Sodium hydrogen phosphate (Na2 HPO4 ), sodium carbonate (Na2 CO3 ), CNBrSepharoseTM Fast Flow (GE17-0981-01), glass columns TricornTM 5/100 (GE28-4064-10) and IgG from bovine serum were purchased from Merck KGaA (Darmstadt, Germany) Hydrochloric acid (HCl) and sodium chloride (NaCl) were purchased from Honeywell Fluka (Seelze, Germany) Lyophilized recombinant protein A (10600-P07E) was purchased from Sino Biological Inc (Eschborn, Germany) Deionized (Mili-Q quality) water was used for all experiments 2.2 Protein A immobilization on polyHIPE 2.4 The pH transition measurements PolyHIPE monoliths with a matrix volume of approximately 0.5 ml were prepared and tested as previously described [41,42] Protein A was dissolved in the immobilization buffer (0.1 M Na2 CO3 /HCl, 0.5 M NaCl, pH 7.4, filtered, 4°C) to a final concentration of 3.46 mg/ml Each polyHIPE sample was placed in the housing [42] and washed with 10 ml of deionized H2 O and 10 ml of immobilization buffer The sample was then removed from the housing, which was washed and dried to remove residual buffer from the surface, leaving only buffer defined with the pore volume in the sample By mixing 86, 132, 234, and 439 μl of protein A solution with the immobilization buffer to a final volume of ml, four different concentrations were prepared Absorbance at 230 nm (Tecan infinite M200pro, Switzerland) was measured to achieve high sensitivity Each protein A solution was pumped through a single polyHIPE sample for hours, to obtain four samples with different amounts of immobilized protein A Again, absorbance was measured at 230 nm After immobilization was completed, the polyHIPE samples were washed extensively with a buffer to remove unbound protein A from pores and then with ml of deionized water to desorb any non-covalently bound protein A No change in absorbance was observed for the deionized water used for washing, indicating that no protein was desorbed from the matrix after immobilization Therefore, the difference in absorbance before and after immobilization is representative to calculate the amount of immobilized protein A, considering initial dilution caused by the buffer present in the matrix pores Finally, samples were washed with 20 mM phosphate/HCl, pH 7.4, deionized water to remove residual non-immobilized protein A and stored in the same phosphate buffer at 4°C Since the maximum buffer capacity determined from the calculated titration curve of protein A corresponds to pH 4.3, this pH was chosen as the initial buffer pH because the highest sensitivity of the pH transition is expected Both acetate and lactate buffer having pKa values of 4.75 and 3.86 [44] have very similar buffer capacity at pH 4.3, therefore it was investigated which buffer provides more pronounced pH transition For this purpose, the polyHIPE sample was measured with buffer A, being either mM acetate buffer, pH 4.3 or mM lactate buffer, pH 4.3, and corresponding buffer B containing in addition 1M NaCl Two CNBrSepharose samples were measured with and mM lactate buffer, pH 4.3 and the corresponding buffer B containing additionally 1M NaCl PolyHIPE samples containing different amounts of immobilized protein A were assayed with mM lactate buffer, while mM lactate buffer and corresponding buffer B containing in addition 1M NaCl, both pH 4.3, were used for the CNBr-Sepharose with different amount of immobilized protein A Buffers were filtered through a 0.22 μm membrane filter and degassed before use All pH transition experiments were performed on an HPLC ÄKTA system at a flow rate of ml/min as described elsewhere [36] After each stepwise change of two buffers, pH equilibrium was reached 2.5 Static binding capacity (SBC) for protein A-Sepharose Protein A-Sepharose samples were first washed three times with the binding buffer (20 mM Tris/HCl at pH 7.4) and always centrifuged in-between 10 ml of a mg/ml IgG solution was added to 0.67 ml of the particles and absorbance at 280 nm was periodically measured The experiment was allowed to proceed for R Mravljak, M Stanticˇ , O Bizjak et al Journal of Chromatography A 1671 (2022) 462976 day at 4°C under gentle mixing The amount of adsorbed IgG was calculated from the difference between the initial and final absorbance, taking into consideration the initial dilution by the buffer present in the particle pores 2.6 Elemental analysis Elemental analysis was performed using a CHNS analyzer (2400 CHNS/O Series II, Perkin Elmer, USA) to determine the nitrogen content in the polyHIPE samples Results and discussion It has already been shown that the pH transition method can accurately measure the amount of ionizable groups on particulate or monolithic supports [36–40] The method is based on the pH change when the matrix of interest undergoes a stepwise change in the buffer composition from high to low ionic strength and vice versa The pH transition represents an intermediate state resulting from the difference in the velocity of retained and non-retained ions as a consequence of the established ion exchange equilibria between ions in solution and on the matrix surface, depending on the dissociation equilibria of the ionizable groups [39,45] The magnitude of the pH change is influenced by the composition and pH of the mobile phase and by the type and amount of ionizable groups on the polymer surface Therefore, the duration and shape of the pH transition can be correlated with the quantity of ionizable groups on the matrix Since the method uses biocompatible buffers, it is less invasive compared to a conventional titration, where strong acids and bases are commonly implemented To investigate whether immobilized protein A can be detected by the pH transition method, monolithic porous polyHIPE polymers with epoxy groups were used [41], so that no further matrix preactivation was required [46] Immobilization of protein A was performed as described in the Materials and methods section, by measuring the absorbance of protein A solution before and after immobilization In addition to the polyHIPE samples containing protein A, a reference polyHIPE sample was also prepared, using the same immobilization protocol but without protein A in the immobilization buffer From the difference in absorbance, it was estimated that 0.25, 0.42, 0.72, and 0.98 mg of protein A was immobilized on the polyHIPE matrix, resulting in immobilized protein A concentrations of 0.48, 0.8, 1.35, and 1.98 mg/ml, respectively No change in absorbance was observed for the polyHIPE reference sample Low concentrations of immobilized protein A were determined, which can be attributed to the low specific surface area of polyHIPEs [42] which limiting the maximum quantity of immobilized protein As samples are therefore were perfectly suited to study the sensitivity of the pH transition method Since the amount of immobilized protein A was determined indirectly by absorbance measurements, we investigated whether direct elemental analysis could be used as well Because the method is destructive, the nitrogen content of all polyHIPE samples was measured only after all other experiments, including pH transition measurements, were completed Unfortunately, no significant difference was found between the samples (including the reference sample) This result indicates that the amount of immobilized protein A was too low to be accurately determined In fact, even for the highest protein A concentration of 1.98 mg/ml, assuming its chemical composition of C1222 H1920 N356 O417 S2 [47], nitrogen accounts for less than 0.17% of the sample mass, which is below the limit of quantification of the instrument [48] Obviously, to estimate such low amounts of immobilized protein A, a very high sensitivity of the pH transition method is required The sensitivity of the method depends on the type of buffer Fig Calculated protein A charge from the amino acid sequence using ProtParam software (Expasy) and the buffer capacity used, its concentration and pH It can be expected that the maximum effect of the immobilized ionizable groups on the pH transition is at the pH at which they exhibit the maximum buffering capacity, namely at their pKa The buffer capacity of Protein A shown in Fig was estimated from the theoretical titration curve generated using ProtParam software (Expasy) based on the amino acid sequence of protein A The buffer capacity of protein A was calculated as the negative difference in the protein charge divided by the change in pH (d[z]/dpH) [49] Three pKa values can be seen from Fig at pH 4.3, 9.8, and 12.4, where the charge of protein A is +11.9, -27.9, and -44.7, respectively To maintain the biological stability of protein A, only the lowest pKa value at pH 4.3 is suitable and was therefore chosen for the pH transition buffers Two biologically compatible buffers, namely acetate (pKa 4.76) and lactate buffer (pKa 3.86) [44], seem to be suitable since they have pKa values close to pH 4.3 As both are formed from monovalent acids, their buffering capacity at pH 4.3 is already much lower than at their maximum, possibly allowing the detection of the pH transition caused by the immobilized protein A On the other hand, even at rather low buffer concentration, the buffer capacity is still sufficient to provide robustness to the measurements Both buffers were tested varying their concentration Despite possible shift in maximum buffer capacity of protein A due to immobilization [50], pH of both buffers was adjusted to 4.3 to investigate if sensitivity of pH transition method is sufficient to detect presence of immobilized protein A Indeed, both buffers showed differences in the pH transition profiles between polyHIPE sample with immobilized protein A and the reference polyHIPE, which were however more pronounced for the lactate buffer (Fig S1) A mM lactate buffer, pH 4.3 was therefore used for further experiments Stepwise change using this buffer was performed on all prepared polyHIPE samples to obtain the pH transition responses shown in Fig Figs 2a and 2b show the pH transition for the stepwise buffer change All pH profiles exhibited a chromatographic peak shape, with its maximum correlating with the amount of immobilized protein A Peaks were analyzed as described in literature [36] by measuring the full width at half maximum (FWHM) and the height of the peaks ( pH) Due to a chromatographic peak shape, better linear correlation [36] was obtained between the pH transition peak height ( pH) and the amount of immobilized protein A plotted in Fig 2c and 2d Since promising results were obtained with polyHIPE samples, the general applicability of the pH transition method for protein A was further studied on particulate CNBr-Sepharose In addition of the higher expected amount of immobilized protein A, due to R Mravljak, M Stanticˇ , O Bizjak et al Journal of Chromatography A 1671 (2022) 462976 Fig pH transition profiles for polyHIPE samples containing (ref), 0.25, 0.42, 0.72, and 0.98 mg immobilized protein A Buffer A: mM lactate buffer, pH 4.3; buffer B: mM lactate buffer containing M NaCl, pH 4.3; flow rate ml/min The pH response for the stepwise change from buffer B to buffer A (a) and buffer A to buffer B (b) Correlation between the pH transition peak height ( pH) and the amount of immobilized protein A for both stepwise changes: buffer B to buffer A (c) and buffer A to buffer B (d) Fig The pH transition response for two columns filled with a particulate Sepharose matrix containing 3.5 and 4.4 mg of protein A, respectively Matrix volume: 0.67 ml; protein A concentration: 5.3 and 6.6 mg/ml pH transition profiles were measured at flow rate of ml/min with lactate buffer, pH 4.3, during a stepwise change: from buffer containing M NaCl to buffer without NaCl (a) and opposite (b) Lactate buffer concentration is shown in graph a higher specific surface area [43], the particulate matrix also allows for higher flexibility in column preparation obtained by mixing particles with different protein A content and varying the matrix volume Initially CNBr-Sepharose was divided into three parts, two of which were immersed in buffer solutions with different protein A concentrations, while one part was immersed in the same buffer solution but without protein A to be used as a reference Again, the absorbance of the solution was measured before and after immobilization and the difference was used to calculate the amount of immobilized protein A (Fig S2), which was estimated to be 5.3 and 6.6 mg/ml, respectively All three matrices were packed into the column (0.67 ml) and the pH transitions were measured during the stepwise change using the same buffer as for polyHIPE samples (Fig 3) Compared to the polyHIPE samples, much longer pH transitions were observed, exceeding 40 ml A longer pH transition was ex- pected due to the higher amount of protein A, but a high pH transition volume is rather impractical to perform routine measurements Since the duration of the pH transition can be tailored by a buffer concentration [36–40], the lactate buffer concentration was increased to mM With this buffer, all pH transitions were completed within 10 ml and although the magnitude of pH excursions decreased, it was still sufficient to accurately detect differences in the amount of immobilized protein A (Fig 3) After the pH transition measurements were completed, matrix containing 5.3 and 6.6 mg/ml of protein A was removed from the columns and mixed to obtain approximately 1.3 ml of matrix containing an average of 5.95 mg/ml of protein A The column was packed with 1.2 ml of this matrix (6.9 mg of protein A), the pH transition was measured, after that the matrix volume was decreased to 0.97 ml (5.7 mg of protein A) and the pH transition was measured again The matrix was removed from the column and mixed with a reference matrix in volume ratios of 0.22/0.77 and R Mravljak, M Stanticˇ , O Bizjak et al Journal of Chromatography A 1671 (2022) 462976 Fig pH transition profiles for a particulate Sepharose matrix bearing (ref), 1.25, 2.5, 3.5, 4.4, 5.7 and 6.9 mg immobilized protein A Buffer A: mM lactate buffer, pH 4.3; buffer B: mM lactate buffer containing M NaCl, pH 4.3; flow rate: ml/min; pH transition peak height ( pH) for the stepwise buffer change from buffer B to buffer A (c) and FWHM for the stepwise buffer change from buffer A to buffer B (d), both correlated to the amount of immobilized protein A 0.38/0.62 One column was filled with 0.92 ml of the first mixture, containing 1.25 mg of protein A, while the other column was filled with 1.1 ml of the second mixture, containing 2.5 mg of protein A pH transitions of both columns were measured This approach allowed us to study pH profiles of different amounts of protein A with a very low protein A matrix volume and to precisely determine the amount of immobilized protein A in each column with respect to the concentration of the original protein A matrix All pH transition profiles are shown in Fig (4a and 4b) When the values obtained from the pH transition profiles were plotted against the amount of immobilized protein A, a good linear correlation was obtained (Fig 4c, d) as previously shown also for polyHIPE samples The only difference was that due to a higher amount of immobilized protein A, resulting in the flat-top pH transition peaks during stepwise change from low to high ionic strength buffer (Fig 4b), FWHM provided better correlation, as discussed elsewhere [36] Because of higher density of immobilized protein A, we were also interested to investigate if the amount of immobilized protein A could also be linearly correlated with the static binding capacity (SBC) towards IgG in this range, since a non-linear trend has been found at high ligand density [34] SBC measurements of matrix bearing 5.3 and 6.6 mg/ml protein A resulted in IgG SBC of 21.8 and 25.0 mg/ml, respectively The amount of adsorbed IgG enabled calculation of ligand utilization, which was 1.22 and 1.09, respectively It can be seen that a ligand utilization above was achieved in both cases, which is in agreement with recent studies [17,18] More importantly, ligand utilization decreased for higher ligand density, a trend previously reported for the dynamic binding capacity of protein A immobilized on monoliths [34] This confirms that SBC cannot be used directly to determine the amount of immobilized protein A when the matrix surface is nearly saturated The reason for lower ligand utilization at higher ligand density are probably steric hindrances [34] due to the large size of IgG compared to a protein A molecule On the other hand, the pH transition method is based on the exchange of ions between the immobilized ligand and the mobile phase, which are very small compared to the size of the protein A ligand Therefore, no hindrances are expected, leading to a linear trend between the pH transition measurements and the amount of immobilized protein A, even at the highest protein A ligand density (Fig 4c, d) As mentioned earlier, changing the buffer concentration affects the sensitivity of the method (Fig 3) Therefore, it can be easily applied to small as well as large chromatographic protein A columns by adjusting the buffer concentration accordingly While the duration of the pH transition is linearly correlated with the reciprocal buffer concentration [38], even larger differences in sensitivity can be obtained by changing the pH value [38], since the buffer capacity is highly non-linearly dependent on pH This allows the extension of this method to chromatographic columns of preparative scale For this reason, the proposed method can be used to evaluate the immobilization process of protein A, but it can also serve as an in-line method for non-invasive monitoring of changes in protein A column performance on analytical or industrial scale On the other hand, it can also be implemented to evaluate immobilization efficiency on microfluidic devices, as it has an even higher sensitivity than potentiometric measurement [27], allowing determination of ligand densities even below 0.5 mg/ml Although the proposed method was tested only on a protein A ligand, due to its flexibility, it can be extended to other immobilized molecules like different proteins, but also to DNA or even nanoparticles bearing ionizable moieties, simply by proper selection of buffer type, concentration and pH value Conclusion A pH transition method based on lactate buffer was developed and implemented to determine the amount of immobilized protein A directly and noninvasively The method is based on a biocompatible buffer typically used in chromatography, therefore its implementation is straightforward and does not require additional equipment Due to its simplicity, it could represent a novel tool for direct, non-invasive determination of the amount of immobilized protein A at laboratory and industrial scale, allowing optimization of ligand utilization during immobilization, but also serving for inprocess monitoring of the protein A stationary phase, even in GMP environment R Mravljak, M Stanticˇ , O Bizjak et al Journal of Chromatography A 1671 (2022) 462976 Author Contributions [16] T.M Pabst, J Thai, A.K Hunter, Evaluation of recent Protein A stationary phase innovations for capture of biotherapeutics, J Chromatogr A 1554 (2018) 45– 60, doi:10.1016/j.chroma.2018.03.060 [17] J Plewka, G.L Silva, R Tscheließnig, H Rennhofer, C Dias-Cabral, A Jungbauer, H.C Lichtenegger, Antibody adsorption in protein-A affinity chromatography in situ measurement of nanoscale structure by small-angle X-ray scattering, J Sep Sci 41 (2018) 4122–4132, doi:10.10 02/jssc.20180 0776 [18] G.L Silva, J Plewka, H Lichtenegger, A.C Dias-Cabral, A Jungbauer, R Tscheließnig, The pearl necklace model in protein A chromatography: Molecular mechanisms at the resin interface, Biotechnol Bioeng 116 (2019) 76–86, doi:10.1002/bit.26843 [19] A.A Shukla, B Hubbard, T Tressel, S Guhan, D Low, Downstream processing of monoclonal antibodies—application of platform approaches, J Chromatogr B 848 (2007) 28–39, doi:10.1016/j.jchromb.2006.09.026 [20] E Müller, J Vajda, Routes to improve binding capacities of affinity resins demonstrated for Protein A chromatography, J Chromatogr B 1021 (2016) 159–168, doi:10.1016/j.jchromb.2016.01.036 [21] X.-H Yang, L.-M Huan, X.-S Chu, Y Sun, Q.-H Shi, A comparative investigation of random and oriented immobilization of protein A ligands on the binding of immunoglobulin G, Biochem Eng J 139 (2018) 15–24, doi:10.1016/j.bej.2018 08.002 [22] J.W Beattie, R.C Rowland-Jones, M Farys, R Tran, S.G Kazarian, B Byrne, Insight into purification of monoclonal antibodies in industrial columns via studies of Protein A binding capacity by in situ ATR-FTIR spectroscopy, Analyst 146 (2021) 5177–5185, doi:10.1039/D1AN00985K [23] B.J Smith, Chemical Cleavage of Proteins, in: New Protein Tech, Humana Press, New Jersey, 1989, pp 71–88, doi:10.1385/0- 89603- 126- 8:71 [24] Y.-P Kim, M.-Y Hong, J Kim, E Oh, H.K Shon, D.W Moon, H.-S Kim, T.G Lee, Quantitative analysis of surface-immobilized protein by TOF-SIMS: Effects of protein orientation and trehalose additive, Anal Chem 79 (2007) 1377–1385, doi:10.1021/ac0616005 [25] M Bonde, H Pontoppidan, D.S Pepper, Direct dye binding—A quantitative assay for solid-phase immobilized protein, Anal Biochem 200 (1992) 195–198, doi:10.1016/0 03- 2697(92)90298- L [26] P Singh, H Morris, A.V Tivanski, A Kohen, Determination of concentration and activity of immobilized enzymes, Anal Biochem 484 (2015) 169–172, doi:10 1016/j.ab.2015.02.014 [27] E Voitechovicˇ , A Korepanov, D Kirsanov, A Legin, Quantification of immobilized protein in pharmaceutical production by bio-assisted potentiometric multisensor system, J Pharm Biomed Anal 150 (2018) 67–71, doi:10.1016/j jpba.2017.11.076 [28] D Wu, R.R Walters, Effects of stationary phase ligand density on highperformance ion-exchange chromatography of proteins, J Chromatogr A 598 (1992) 7–13, doi:10.1016/0021-9673(92)85108-6 [29] H.W Robinson, C.G Hogden, The biuret reaction in the determination of serum proteins, J Biol Chem 135 (1940) 707–725, doi:10.1016/S0021-9258(18) 73134-7 [30] O Lowry, N Rosebrough, A.L Farr, R Randall, Protein measurement with the Folin phenol reagent, J Biol Chem 193 (1951) 265–275, doi:10.1016/ S0021- 9258(19)52451- [31] T Basinska, S Slomkowski, The direct determination of protein concentration for proteins immobilized on polystyrene microspheres, J Biomater Sci Polym Ed (1992) 115–125, doi:10.1163/156856291×00223 [32] M.M Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal Biochem 72 (1976) 248–254, doi:10.1016/0 03- 2697(76)90527- [33] P.K Smith, R.I Krohn, G.T Hermanson, A.K Mallia, F.H Gartner, M.D Provenzano, E.K Fujimoto, N.M Goeke, B.J Olson, D.C Klenk, Measurement of protein using bicinchoninic acid, Anal Biochem 150 (1985) 76–85, doi:10.1016/ 0 03- 2697(85)90442- ˇ [34] U Cernigoj, U Vidic, B Nemec, J Gašperšicˇ , J Vidicˇ , N Lendero Krajnc, A Štrancar, A Podgornik, Characterization of methacrylate chromatographic monoliths bearing affinity ligands, J Chromatogr A 1464 (2016) 72–78, doi:10 1016/j.chroma.2016.08.014 [35] A Podgornik, T.B Tennikova, Chromatographic Reactors Based on Biological Activity, Adv Biochem Eng Biotechnol (2002) 165–210, doi:10.1007/ 3- 540- 45345- 8_5 [36] R Mravljak, O Bizjak, P Krajnc, M Paljevac, A Podgornik, Non-invasive determination of ionizable ligand group density on high internal phase emulsion derived polymer, J Chromatogr A 1652 (2021) 462077, doi:10.1016/j.chroma 2021.462077 [37] N Lendero, J Vidicˇ , P Brne, A Podgornik, A Štrancar, Simple method for determining the amount of ion-exchange groups on chromatographic supports, J Chromatogr A 1065 (2005) 29–38, doi:10.1016/j.chroma.2004.10.072 [38] N Lendero, J Vidicˇ , P Brne, V Frankovicˇ , A Štrancar, A Podgornik, Characterization of ion exchange stationary phases via pH transition profiles, J Chromatogr A 1185 (2008) 59–70, doi:10.1016/j.chroma.2008.01.023 [39] T.M Pabst, G Carta, pH transitions in cation exchange chromatographic columns containing weak acid groups, J Chromatogr A 1142 (2007) 19–31, doi:10.1016/j.chroma.2006.08.066 [40] T.E Bankston, L Dattolo, G Carta, pH Transients in hydroxyapatite chromatography columns-Experimental evidence and phenomenological modeling, J Chromatogr A 1217 (2010) 2123–2131, doi:10.1016/j.chroma.2010.02.004 [41] R Mravljak, O Bizjak, B Božicˇ , M Podlogar, A Podgornik, Flow-through PolyHIPE silver-based catalytic reactor, Polymers (Basel) 13 (2021) 880, doi:10 3390/polym13060880 Rok Mravljak performed immobilization and static binding capacity measurements, partially pH transition experiments and analyzed data, contributed to the manuscript, Metka Stanticˇ also performed immobilization experiments, Ožbej Bizjak performed pH transition experiments, Aleš Podgornik defined the goal of the work, contributing to experimental design, prepared the manuscript draft and finalized the manuscript Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper Acknowledgment The financial support is gratefully acknowledged from the Slovenian Research Agency (ARRS) through project J2-9440 and program P1-0153 We want to thank Rok Ambrožicˇ for valuable discussions Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.chroma.2022.462976 References [1] J.C.S dos Santos, O Barbosa, C Ortiz, A Berenguer-Murcia, R.C Rodrigues, R Fernandez-Lafuente, Importance of the Support Properties for Immobilization or Purification of Enzymes, ChemCatChem (2015) 2413–2432, doi:10 1002/cctc.201500310 ´ A Podgornik, Appli[2] M Stanticˇ , G Guncˇ ar, D Kuzman, R Mravljak, T Cvijic, cation of lectin immobilized on polyHIPE monoliths for bioprocess monitoring of glycosylated proteins, J Chromatogr B 1174 (2021) 122731, doi:10.1016/j jchromb.2021.122731 [3] G Tripodo, G Marrubini, M Corti, G Brusotti, C Milanese, M Sorrenti, L Catenacci, G Massolini, E Calleri, Acrylate-based poly-high internal phase emulsions for effective enzyme immobilization and activity retention: from computationally-assisted synthesis to pharmaceutical applications, Polym Chem (2018) 87–97, doi:10.1039/C7PY01626C [4] S.K Vashist, E.Marion Schneider, E Lam, S Hrapovic, J.H.T Luong, One-step antibody immobilization-based rapid and highly-sensitive sandwich ELISA procedure for potential in vitro diagnostics, Sci Rep (2015) 4407, doi:10.1038/ srep04407 [5] D Kim, A.E Herr, Protein immobilization techniques for microfluidic assays, Biomicrofluidics (2013) 041501, doi:10.1063/1.4816934 [6] A Ananth, M Genua, N Aissaoui, L Díaz, N.B Eisele, S Frey, C Dekker, R.P Richter, D Görlich, Reversible immobilization of proteins in sensors and solid-state nanopores, Small 14 (2018) 1703357, doi:10.1002/smll.201703357 [7] K.M Łacki, ˛ F.J Riske, Affinity Chromatography: An Enabling Technology for Large-Scale Bioprocessing, Biotechnol J 15 (2020) 1800397, doi:10.1002/biot 201800397 [8] J Scheffel, S Kanje, J Borin, S Hober, Optimization of a calcium-dependent Protein A-derived domain for mild antibody purification, MAbs 11 (2019) 1492–1501, doi:10.1080/19420862.2019.1662690 [9] V Amritkar, S Adat, V Tejwani, A Rathore, R Bhambure, Engineering Staphylococcal Protein A for high-throughput affinity purification of monoclonal antibodies, Biotechnol Adv 44 (2020) 107632, doi:10.1016/j.biotechadv.2020 107632 [10] Q Shi, Y Sun, Protein A-based ligands for affinity chromatography of antibodies, Chinese J Chem Eng 30 (2021) 194–203, doi:10.1016/j.cjche.2020.12.001 [11] J Scheffel, S Hober, Highly selective Protein A resin allows for mild sodium chloride-mediated elution of antibodies, J Chromatogr A 1637 (2021) 461843, doi:10.1016/j.chroma.2020.461843 [12] R Hahn, Comparison of protein A affinity sorbents, J Chromatogr B 790 (2003) 35–51, doi:10.1016/S0021- 9673(02)02003- [13] R Hahn, P Bauerhansl, K Shimahara, C Wizniewski, A Tscheliessnig, A Jungbauer, Comparison of protein A affinity sorbents, J Chromatogr A 1093 (2005) 98–110, doi:10.1016/j.chroma.2005.07.050 [14] R Hahn, K Shimahara, F Steindl, A Jungbauer, Comparison of protein A affinity sorbents III Life time study, J Chromatogr A 1102 (2006) 224–231, doi:10.1016/j.chroma.2005.10.083 [15] S Ghose, B Hubbard, S.M Cramer, Binding capacity differences for antibodies and Fc-fusion proteins on protein A chromatographic materials, Biotechnol Bioeng 96 (2007) 768–779, doi:10.1002/bit.21044 R Mravljak, M Stanticˇ , O Bizjak et al Journal of Chromatography A 1671 (2022) 462976 [42] R Mravljak, O Bizjak, M Podlogar, A Podgornik, Effect of polyHIPE porosity on its hydrodynamic properties, Polym Test 93 (2021) 106590, doi:10.1016/j polymertesting.2020.106590 [43] Cytiva, CNBr-activated Sepharose 4B affinity media instructions for use table of contents, (2020) https://cdn.cytivalifesciences.com/dmm3bwsv3/AssetStream aspx?mediaformatid=10061&destinationid=10016&assetid=12981 [44] R.J Prankerd, Critical compilation of pKa values for pharmaceutical substances, in: H.G Brittain (Ed.), Profiles Drug Subst Excip Relat Methodol., volume 33, Academic Press, 2007: p 726 [45] F.G Helfferich, B.J Bennett, Weak electrolytes, polybasic acids, and buffers in anion exchange columns I Sodium acetate and sodium carbonate systems, React Polym Ion Exch Sorbents (1984) 51–66, doi:10.1016/0167-6989(84) 90122-3 [46] P Krajnc, N Leber, D Štefanec, S Kontrec, A Podgornik, Preparation and characterisation of poly(high internal phase emulsion) methacrylate monoliths and their application as separation media, J Chromatogr A 1065 (2005) 69–73, doi:10.1016/j.chroma.2004.10.051 [47] C Lendel, J Dogan, T Härd, Structural Basis for Molecular Recognition in an Affibody:Affibody Complex, J Mol Biol 359 (2006) 1293–1304, doi:10.1016/j jmb.2006.04.043 [48] PerkinElmer, 2400 Series II CHNS/O elemental analysis, perkin elmer (2011) https://www.perkinelmer.com/lab-solutions/resources/docs/BRO_2400_ SeriesII_CHNSO_Elemental_Analysis.pdf [49] H Yamano, K Miyagawa, Buffer capacity curves of green tea extracts using a personal computer with numerically treated online software, Food Sci Technol Int Tokyo (1997) 69–73, doi:10.3136/fsti9596t9798.3.69 [50] F Secundo, Conformational changes of enzymes upon immobilisation, Chem Soc Rev 42 (2013) 6250, doi:10.1039/c3cs35495d ... vol.% acetone as a tracer and a 30 μl loop using HPLC system (ÄKTAExplorer, GE Healthcare, Uppsala, Sweden) at a flow rate of ml/min Materials and methods 2.1 Chemicals Acetic acid, lactic acid,... capacity of Protein A shown in Fig was estimated from the theoretical titration curve generated using ProtParam software (Expasy) based on the amino acid sequence of protein A The buffer capacity... non-invasive determination of the amount of immobilized protein A at laboratory and industrial scale, allowing optimization of ligand utilization during immobilization, but also serving for inprocess