Analytical characterization of complex, biotechnological feedstocks by pH gradient ion exchange chromatography for purification process development

Kröner, F.; Hanke, Alexander T.; Nfor, Beckley K.; Pinkse, Martijn W. H.; Verhaert, Peter; Ottens, Marcel; Hubbuch, Jürgen

doi:10.1016/j.chroma.2013.08.034

Cited by 16 publications

(13 citation statements)

References 30 publications

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“…6, nearly all the unwanted components had already been removed by highly efficient IEX step, and the purity of rG17PE38 could reach about 94%. Also, nucleic acids and endotoxin in the sample may be eliminated from the target protein through IEX [17].…”

Section: Discussionmentioning

confidence: 99%

Establishment of a three-step purification scheme for a recombinant protein rG17PE38 and its characteristics identification

Xia

Liu

et al. 2015

Journal of Chromatography B

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Section: Discussionmentioning

confidence: 99%

Establishment of a three-step purification scheme for a recombinant protein rG17PE38 and its characteristics identification

Xia

Liu

et al. 2015

Journal of Chromatography B

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“…Recently developed linear pH gradients should be used to gain more information about the elution pH of HCPs and target proteins [35, 36], helping to define the conditions that must be screened for particular separation problems (including intermediate purification) and making the use of potentially misleading indicators such as the p I obsolete (Fig. 3F).…”

Section: Discussionmentioning

confidence: 99%

Generic chromatography‐based purification strategies accelerate the development of downstream processes for biopharmaceutical proteins produced in plants

Buyel

Fischer

2014

Biotechnology Journal

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Plants offer a valuable alternative to cultured mammalian cells for the production of recombinant biopharmaceutical proteins. However, the target protein typically represents only a minor fraction of the total protein in the initial plant extract, which means that the development of product-specific chromatography-based purification strategies is often laborious and expensive. To address this challenge, we designed a generic downstream process that is suitable for the purification of recombinant proteins with diverse properties from plant production platforms. This was achieved by focusing on the binding behavior of tobacco host cell proteins (HCPs) to a broad set of chromatography resins under different pH and conductivity conditions. Strong cation exchanger and salt-tolerant anion exchanger resins exhibited the best resolution of tobacco HCPs among the 13 tested resins, and their selectivity was easy to manipulate through the adjustment of pH and conductivity. The advantages, such as direct capture of a target protein from leaf extract, and limitations, such as low binding capacity, of various chromatography ligands and resins are discussed. We also address the most useful applications of the chromatography ligands, namely recovery of proteins with a certain pI, in a downstream process that aims to purify diverse plant-derived biopharmaceutical proteins. Based on these results, we describe generic purification schemes that are suitable for acidic, neutral, and basic target proteins, as a first step toward the development of industrial platform processes.

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“…A long-range cation exchange gradient chromatofocusing step carried out on an Äkta Explorer 10 system equipped with a Mono S 4.6/100 column (GE-Healthcare, Uppsala, Sweden) according to a protocol described elsewhere [29] served as the first separation dimension. A total of 96 fractions of 200 l were collected at regular intervals over the course of the gradient.…”

Section: Experimental Apparatus and Proceduresmentioning

confidence: 99%

Fourier transform assisted deconvolution of skewed peaks in complex multi-dimensional chromatograms

Hanke

Verhaert

Wielen

et al. 2015

Journal of Chromatography A

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Analytical characterization of complex, biotechnological feedstocks by pH gradient ion exchange chromatography for purification process development

Cited by 16 publications

References 30 publications

Establishment of a three-step purification scheme for a recombinant protein rG17PE38 and its characteristics identification

Establishment of a three-step purification scheme for a recombinant protein rG17PE38 and its characteristics identification

Generic chromatography‐based purification strategies accelerate the development of downstream processes for biopharmaceutical proteins produced in plants

Fourier transform assisted deconvolution of skewed peaks in complex multi-dimensional chromatograms

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