Research article

Impact of operating conditions on chromatographic column performance: experimental studies on adsorption of high-value minor whey proteins

  • Received: 11 January 2017 Accepted: 02 March 2017 Published: 15 March 2017
  • Over the last decade, ion-exchange chromatography (IEC) has been extensively explored for protein purification at both small and large scales. Despite several IEC columns are commercialized, the physical phenomena underlying the adsorption of proteins on ion-exchange columns performance has not been thoroughly investigated. In this work, the influence of operating conditions on the adsorption of lactoperoxidase (LP) and lactoferrin (LF) on cation exchange chromatography adsorbent is experimentally studied in order to understand fundamental pertaining to underlying mechanism. Analysis was carried out in columns with different IDs (7.7 and 16 mm), packed for 100 mm with 90 μm particle size polymer-grafted cation exchanger. The flow distribution was measured using acetone as a non-binding tracer. An evaluation of van Deemter plots was done as well as LP breakthrough curves at different flow rates and LP loading concentrations. The results were compared with two columns in terms of efficiency and the LP binding capacity. The dynamic binding capacity at 10% breakthrough was found to be independent of the applied flow rate. Surprisingly for both systems, LP breakthrough takes place later at higher loading concentrations, which is in contrast to IEC. The results propose a major presence of non-ideal effects as steric shielding and charge repulsion of protein in the adsorption. In addition, the accessibility of binding sites for protein at higher concentrations seems more available than sodium counter-ions in buffer.

    Citation: Naeimeh Faraji, Yan Zhang, Ajay K. Ray. Impact of operating conditions on chromatographic column performance: experimental studies on adsorption of high-value minor whey proteins[J]. AIMS Bioengineering, 2017, 4(2): 223-238. doi: 10.3934/bioeng.2017.2.223

    Related Papers:

  • Over the last decade, ion-exchange chromatography (IEC) has been extensively explored for protein purification at both small and large scales. Despite several IEC columns are commercialized, the physical phenomena underlying the adsorption of proteins on ion-exchange columns performance has not been thoroughly investigated. In this work, the influence of operating conditions on the adsorption of lactoperoxidase (LP) and lactoferrin (LF) on cation exchange chromatography adsorbent is experimentally studied in order to understand fundamental pertaining to underlying mechanism. Analysis was carried out in columns with different IDs (7.7 and 16 mm), packed for 100 mm with 90 μm particle size polymer-grafted cation exchanger. The flow distribution was measured using acetone as a non-binding tracer. An evaluation of van Deemter plots was done as well as LP breakthrough curves at different flow rates and LP loading concentrations. The results were compared with two columns in terms of efficiency and the LP binding capacity. The dynamic binding capacity at 10% breakthrough was found to be independent of the applied flow rate. Surprisingly for both systems, LP breakthrough takes place later at higher loading concentrations, which is in contrast to IEC. The results propose a major presence of non-ideal effects as steric shielding and charge repulsion of protein in the adsorption. In addition, the accessibility of binding sites for protein at higher concentrations seems more available than sodium counter-ions in buffer.


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    [1] Fritz JS (2004) Early milestones in the development of ion-exchange chromatography: a personal account. J Chromatogr A 1039: 3–12. doi: 10.1016/j.chroma.2003.12.068
    [2] Atamna IZ, Muschick GM, Issaq HJ (1989) The effect of column diameter on HPLC separations using constant length columns. J Liq Chromatogr R T 12: 258–298.
    [3] Gritti F, Guiochon G (2012) Theoretical and experimental impact of the bed aspect ratio on the axial dispersion coefficient of columns packed with 2.5 μm particles. J Chromatogr A 1262: 107–121. doi: 10.1016/j.chroma.2012.09.001
    [4] Rathore SA, Velayudhan A (2002) An overview of scale-up in preparative chromatography in: scale-up and optimization in preparative chromatography: principles and biopharmaceutical applications, Marcel Dekker, New York.
    [5] Rathore AS, Velayudhan A (2003) Guidelines for optimization and scale-up in preparative chromatography. Biopharm Int 16: 34–42.
    [6] Levison PR, Badger SE, Toome DW (1992) Economic considerations important in the scale-up of an ovalbumin separation from hen egg-white on the anion exchange cellulose DE92. J Chromatogr A 590: 49–58. doi: 10.1016/0021-9673(92)87005-S
    [7] Gerberding SJ, Byers CH (1998) Preparative ion-exchange chromatography of proteins from dairy whey. J Chromatogr A 808: 141–151. doi: 10.1016/S0021-9673(98)00103-4
    [8] Pedersen L, Mollerup J (2000) Scale-up of chromatographic separations in ion-exchange chromatography, Poster presented at ISPPP 2000, Ljubljana, Slovenia, 5–8.
    [9] Pedersen L, Mollerup J (2001) Scale-up of chromatographic separations in ion-exchange chromatography, Poster presented at PREP' 2001, Washington, DC, USA, 26–29.
    [10] Pedersen L, Mollerup J, Hansen E, et al. (2003) Whey proteins as a model system for chromatographic separation of proteins. J Chromatogr B 790: 161–173. doi: 10.1016/S1570-0232(03)00127-2
    [11] Lightfoot EN (1999) Speeding the design of bioseparations: a heuristic approach to engineering design. Ind Eng Chem Res 38: 3628–3634. doi: 10.1021/ie9900566
    [12] Lenhoff AM (1987) Significance and estimation of chromatographic parameters. J Chromatogr A 384: 285–299.
    [13] Stanley BJ, Savage TL, Geraghty J (1998) Calculation of the hydrodynamic contribution to peak asymmetry in high-perfomance liquid chromatography using the equilibrium-dispersive model. Anal Chem 70: 1610–1617. doi: 10.1021/ac971096r
    [14] Levison PR (2003) Large-scale ion-exchange column chromatography of proteins comparison of different formats. J Chromatogr B 790: 17–33. doi: 10.1016/S1570-0232(03)00087-4
    [15] Guiochon G, Felinger A, Shirazi DG, et al. (2006) Fundamentals of preparative and nonlinear chromatography, 2nd ed., Elsevier Academic Press, New York.
    [16] Whitley RD, Zhang X, Wang NHL (1994) Protein denaturation in nonlinear isocratic and gradient elution chromatography. Aiche J 40: 1067–1081. doi: 10.1002/aic.690400617
    [17] Kaltenbrunner O, Jungbauer A, Yamamoto S (1997) Prediction of the preparative chromatography performance with a very small column. J Chromatogr A 760: 41–53. doi: 10.1016/S0021-9673(96)00689-9
    [18] Li P, Xiu G, Rodrigues AE (2004) Modeling breakthough and elution curves in fixed bed of inert core adsorbents: analytical and approximate solutions. Chem Eng Sci 59: 3091–3103. doi: 10.1016/j.ces.2004.04.034
    [19] Jacobson N, Degerman M, Stenborg E, et al. (2007) Model based robustness analysis of an ion-exchange chromatography step. J Chromatogr A 1138: 109–119. doi: 10.1016/j.chroma.2006.10.057
    [20] Schneiderman S, Varadaraju H, Zhang L, et al. (2011) Mathematical model using non-uniform flow distribution for dynamic protein breakthrough with membrane adsorption media. J Chromatogr A 1218: 9121–9127. doi: 10.1016/j.chroma.2011.10.063
    [21] Van Deemter JJ, Zuiderweg FJ, Klinkenberg A (1956) Longitudinal diffusion and resistance to mass transfer as causes of nonideality in chromatography. Chem Eng Sci 5: 271–289. doi: 10.1016/0009-2509(56)80003-1
    [22] Rodrigues AE (1993) An extended van Deemter equation (Rodrigues equation) for performing chromatographic processes using large-pore, permeable particles, 6: 20.
    [23] Rodrigues AE, Loureiro JM, Chenou C (1995) Bioseparations with permeable particles. J Chromatogr B 664: 233–240. doi: 10.1016/0378-4347(94)00361-8
    [24] Patel KD, Jerkovich AD, Link JC, et al. (2004) In-depth characterization of slurry packed capillary columns with 1.0 µm nonporous particles using reversed-phase isocratic ultrahigh-pressure liquid chromatography. Anal Chem 76: 5777–5786.
    [25] Van Beijeren P, Kreis P, Zeiner T (2012) Ion exchange membrane adsorption of bovine serum albumin: impact of operating and buffer conditions on breakthrough curves. J Membr Sci s415–s416: 568–576.
    [26] Harinarayan C, Mueller J, Ljunglöf A, et al. (2006) An exclusion mechanism in ion exchange chromatography. Biotechnol Bioeng 95: 775–787. doi: 10.1002/bit.21080
    [27] Billakanti JM, Fee CJ (2009) Characterization of cryogel monoliths for extraction of minor proteins from milk by cation exchange. Biotechnol Bioeng 103: 1155–1163. doi: 10.1002/bit.22344
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