pH-gradient ion-exchange chromatography: An analytical tool for design and optimization of protein separations

Ahamed, Tangir; Nfor, Beckley K.; Verhaert, Peter; Dedem, G.W.K. van; Wielen, Luuk A.M. van der; Eppink, M.H.M.; Sandt, Emile J.A.X. van de; Ottens, Marcel

doi:10.1016/j.chroma.2007.07.010

Cited by 65 publications

(55 citation statements)

References 28 publications

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“…by running the chromatographic experiments at pH 10 with (strong) anion exchangers or at pH 3 with (strong) cation exchangers. This experimental design could clearly influence chromatographic protein behaviour making the separation much more sensitive to the shape of the titration curves, as reported previously [9].…”

Section: Correlating Proteomic Routines With Chromatographic Behaviourmentioning

confidence: 66%

“…Therefore, little correlation was observed between pI values and elution pH in such , nonnative' systems [11]. Moreover, pI values for neutral proteins are also not correlated to elution behaviour during pH-gradient chromatography [9].…”

Section: Introductionmentioning

confidence: 98%

“…Traditionally, experimental techniques like IEF and, especially, electrophoretic titration curve analysis have been employed to anticipate optimal IEX chromatographic conditions [8]. Recently, pH-gradient ionexchange chromatography has also been proposed [9]. Computational approaches were also undertaken to predict protein separations onto ion exchangers [10].…”

Section: Introductionmentioning

confidence: 99%

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Tailoring orthogonal proteomic routines to understand protein separation during ion exchange chromatography

Cabrera¹,

Zhelyazkova²,

Galvis³

et al. 2008

J of Separation Science

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Original PaperTailoring orthogonal proteomic routines to understand protein separation during ion exchange chromatography Surface charge, molecular weight, and folding state are known to influence protein chromatographic behaviour onto ion exchangers. Experimentally, information related to such factors can be gathered via 2-DE methods. The application of 2-D PAGE under denaturing/reducing conditions was already shown to reveal separation trends within a large protein population from cell extracts. However, ion-exchange chromatography normally runs under native conditions. A tailored protocol consisting in a first separation based on IEF on Immobiline TM strips under native conditions followed by a second dimension SDS-PAGE run was adopted. The chromatographic versus electrophoretic separation behaviours of two model proteins, thaumatin (TAU) and BSA, were compared to better understand which proteomic routine would be better suited to anticipate IEX chromatographic separations. It was observed that the information contained in the pI value obtained with the adapted 2-DE protocol showed better correlation with the IEX chromatographic behaviour. On the other hand, chromatographic separations performed in the presence of urea as a denaturant have demonstrated the potential influence of hydrodynamic radius/ conformation on protein separation. Moreover, the information provided by such 2-D system correlated well with the chromatographic behaviour of an additional set of pure proteins. An initial prediction of protein ion-exchange chromatographic behaviour could be possible utilizing an experimental approach based on 2-DE running under milder chemical conditions. This technique provides information that more closely resembles the separation behaviour observed with a complex biotechnological feedstock.

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Section: Correlating Proteomic Routines With Chromatographic Behaviourmentioning

confidence: 66%

Section: Introductionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Tailoring orthogonal proteomic routines to understand protein separation during ion exchange chromatography

Cabrera¹,

Zhelyazkova²,

Galvis³

et al. 2008

J of Separation Science

View full text Add to dashboard Cite

show abstract

“…It was recently demonstrated that pH gradient ion-exchange chromatography can be applied effectively as an analytical tool for the fractionation and characterization of crude protein mixtures and for pH optimization of an ionexchange step. 69 Thermodynamic generalization was proposed as a way of interrelating the relevant thermodynamic properties of proteins in order to reduce the number model parameters and hence experimentation required for modeling the purification units involved. 68 A rational synthesis methodology is required for rapidly generating alternative process options and for screening them to select the best one.…”

Section: Hybrid Methodsmentioning

confidence: 99%

Design strategies for integrated protein purification processes: challenges, progress and outlook

Nfor

Ahamed

Dedem

et al. 2007

J of Chemical Tech & Biotech

Self Cite

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The key to successful and efficient protein purification is the selection of the most appropriate purification techniques and their combination in a logical way to obtain the desired purification in the minimum number of steps. However, the rationalization of protein purification process development is faced with a number of challenges. In this paper, the challenges in protein purification process development are captured. The state-ofthe-art in protein purification process synthesis and design is reviewed and the strengths and weaknesses of the current strategies highlighted. Finally, views on the future directions of protein purification process development are presented.

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“…The pI values of each of the five model proteins were calculated from their primary structure. The protein sequence and pI calculation were taken from the ProtParam tool available on the ExPASy Proteomics Server (http://www.expasy.org) [20,21]. As shown in Table 1, three of the five computed values follow the elution pH values obtained from the chromatographs and two did not closely follow the maximum eluting pH.…”

Section: Chromatographic Behavior Of Model Proteinsmentioning

confidence: 99%

pH‐based cation exchange chromatography in the capture and elution of monoclonal antibodies

Snyder

2011

J of Separation Science

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Cation exchange chromatography separates adsorbed proteins by controlling the salt concentration or the mobile phase pH. This study examines the pH-based method for binding and elution of monoclonal antibodies (MAbs). Five different clones with isoelectric points from 6 to 9 were evaluated. We performed our studies using a new chromatography resin (Nuvia™ S), which has high binding capacity. A three-column process incorporating Nuvia S as a capture step was also demonstrated for the purification of MAb from tissue culture fluid. Chromatography performance of Nuvia S was demonstrated in a 50-cycle study.

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pH-gradient ion-exchange chromatography: An analytical tool for design and optimization of protein separations

Cited by 65 publications

References 28 publications

Tailoring orthogonal proteomic routines to understand protein separation during ion exchange chromatography

Tailoring orthogonal proteomic routines to understand protein separation during ion exchange chromatography

Design strategies for integrated protein purification processes: challenges, progress and outlook

pH‐based cation exchange chromatography in the capture and elution of monoclonal antibodies

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