Efficient purification of recombinant proteins fused to maltose-binding protein by mixed-mode chromatography

Cabanne, Charlotte; Pezzini, J.; Joucla, Gilles; Hocquellet, Agnès; Barbot, C.; Garbay, Bertrand; Santarelli, Xavier

doi:10.1016/j.chroma.2009.03.048

Cited by 20 publications

(7 citation statements)

References 23 publications

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“…It is reported that multimodal chromatographic resins could be used as efficient substitutes to classical purification matrices such as affinity and hydrophobic interaction chromatography for the purification of mAbs and other proteins, including serum albumin, penicillin acylase or maltose binding protein (MBP)‐tagged proteins . In a previous study by the present authors, electropositive multimodal Capto adhere and its improved version of Capto adhere ImpRes were found to constitute an efficient antibody processing step, especially in the removal of HMW aggregates …”

Section: Resultsmentioning

confidence: 77%

A seamless and nonprotein A‐based monoclonal antibody purification platform using void‐exclusion anion exchange chromatography and electropositive multimodal chromatography enhanced by advance chromatin extraction

Liu

Chen

Fan

et al. 2019

J of Chemical Tech & Biotech

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BACKGROUND Downstream production of monoclonal antibodies (mAbs) involves several complex unit operations, directly affecting whether they can be used in clinical practice. Protein A affinity chromatography is considered the de facto standard due to its ability to selectively capture antibodies from cell culture supernatant (CCS). Recent studies on various associations of host cell contaminants have shown that downstream purification can be enhanced substantially with advance chromatin removal, facilitating the development of more promising nonaffinity purification platforms. RESULTS In this study, void‐exclusion anion exchange chromatography (VEAX) was systematically characterized for its potential use as an initial antibody separation and recovery step. Void exclusion refers to a new chromatographic mode, which is different from the traditional bind‐elute and flow‐through operations. The new separation method is based on the size of the molecules as well as their surface charges. This process causes the initial immunoglobulin G (IgG) separation to become independent of sample composition (especially for pH and conductivity), eliminating the need to equilibrate the sample. Electropositive multimodal chromatography was then seamlessly incorporated with direct protein loading for further antibody polishing. With the aid of advanced chromatin removal, the two‐step purification platform achieved production of an antibody with <1 ppb DNA, <1 ppm HCP and <0.05% high‐molecular‐weight aggregates with IgG recovery of 90.4%. CONCLUSION This outcome provides a highly promising nonaffinity purification process and a competitive alternative for the current protein A‐dominated antibody processing strategy. © 2019 Society of Chemical Industry

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Section: Resultsmentioning

confidence: 77%

A seamless and nonprotein A‐based monoclonal antibody purification platform using void‐exclusion anion exchange chromatography and electropositive multimodal chromatography enhanced by advance chromatin extraction

Liu

Chen

Fan

et al. 2019

J of Chemical Tech & Biotech

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“…The adsorbed enzyme could not be eluted and no catalase activity was recovered under any of the elution conditions studied. As catalase is inactive below pH 4.5, we did not endeavour to use lower pH to recover the active protein, although reports prove lowering of pH below 4.5 favours weakening of protein interaction with the PPA ligand (Brochier et al, 2008;Brochier et al, 2009;Cabanne et al, 2009).…”

Section: Effect Of Different Buffers On Adsorption/desorption Of Blacmentioning

confidence: 99%

“…These mixed-mode ligands are recently gaining importance as the choice of sorbents for purification of many industrially important recombinant proteins Cabanne et al, 2009;Pezzini et al, 2009), milk protein (Brochier et al, 2008), polyclonal antibody (Ranjini et al, 2010) and monoclonal antibody (Toueille et al, 2011). All these reports show that these sorbents could be effectively used at the primary capture or intermediate purification step.…”

Section: Introductionmentioning

confidence: 99%

Study of catalase adsorption on two mixed‐mode ligands and the mechanism involved therein

Ranjini

Vijayalakshmi

2012

J of Molecular Recognition

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Mixed-mode chromatography sorbents n-hexylamine HyperCel™ (HEA) and phenylpropylamine HyperCel™ (PPA) were evaluated for the study of adsorption of catalase from two different sources. Various parameters such as buffer composition, ionic strength and pH were investigated to study the mechanism of interaction of commercially available pre-purified catalase from Bovine liver, purified catalase from black gram (Vigna mungo) and crude extract of black gram containing catalase with these mixed-mode ligands. A simple and economical screening protocol for identifying optimal buffer conditions for adsorption and desorption of catalase was established with micro volumes of the sorbent in batch mode. With HEA HyperCel, it was observed that pre-purified catalase from both bovine liver and black gram was completely retained at pH 7.0, irrespective of the presence or absence of NaCl in the adsorption buffer, whereas the catalase from crude extract of black gram was completely retained only in the presence of 0.2 M salt in the adsorption buffer. The elution of catalase from both the sources was accomplished by lowering the pH to 4.5 in absence of salt. In case of PPA HyperCel, catalase from both the sources was very strongly adsorbed under different buffer conditions studied, and elution did not yield a significant catalase activity. From the screening experiments, it could be concluded that the interaction of catalase with HEA HyperCel could be dominated by hydrophobic forces with minor contributions from ionic interaction and with PPA HyperCel, it could be a combination of different non-covalent interactions acting on different loci on the surface of the protein.

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“…The salt‐tolerant adsorption properties of HEA and PPA HyperCel resins were successfully utilized to purify recombinant allergen (rBet v 1a) and achieve ninefold purification from “physiological‐like” crude extract . In another example, the combined action of the same two resins was superior to affinity purification of maltose‐binding fusions from E. coli extracts . Because of complex interactions of proteins with mixed‐modal ligands, the screening of a wide range of conditions is often required to comprehensively evaluate ligand–protein interaction and identify the design space for process development …”

Section: Introductionmentioning

confidence: 99%

“…20 In another example, the combined action of the same two resins was superior to affinity purification of maltose-binding fusions from E. coli extracts. 21 Because of complex interactions of proteins with mixed-modal ligands, the screening of a wide range of conditions is often required to comprehensively evaluate ligand-protein interaction and identify the design space for process development. [22][23][24][25] In this study, we used high-throughput screening (HTS) and batch adsorption experiments to obtain a better understanding of OPN interaction with three mixed-modal resins (Capto adhere, HEA HyperCel, and PPA HyperCel) and select optimal adsorption and elution conditions.…”

Section: Introductionmentioning

confidence: 99%

Exploring the separation power of mixed‐modal resins for purification of recombinant osteopontin from clarified Escherichia coli lysates

Guo

Ravi

Mayfield

et al. 2018

Biotechnology Progress

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Osteopontin (OPN) is a structural protein with potential value in therapeutic and diagnostic applications. Low titer, acidic isoelectric point, and the lack of well‐defined secondary and tertiary structure were some of the challenges that complicated purification development of OPN from recombinant Escherichia coli lysates. Reported processes for OPN recovery from recombinant sources use nonorthogonal unit operations and often suffer from low yield. In this work, we expanded the search for an optimal OPN purification method by including mixed‐modal resins with both ionic and hydrophobic properties (Capto adhere, HEA HyperCel, and PPA HyperCel). Plate‐based high‐throughput screening (HTS) platform revealed useful information about the interactions between the three different ligands and OPN as function of pH and ionic strength. The HTS data allowed the selection of OPN adsorption and elution conditions that were tested and optimized in a batch mode. In terms of purification factor and yield, HEA HyperCel performed significantly better than the other two mixed‐modal resins. Pairing HEA HyperCel with a strong anion exchange step (Capto Q) resulted in a two‐step purification process that achieved 45‐fold purification of OPN with a final purity of 95% and 44% overall yield. The orthogonality provided by mixed‐modal and ion exchange steps resulted in higher yield in fewer unit operations than reported processes. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2722, 2019

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Efficient purification of recombinant proteins fused to maltose-binding protein by mixed-mode chromatography

Cited by 20 publications

References 23 publications

A seamless and nonprotein A‐based monoclonal antibody purification platform using void‐exclusion anion exchange chromatography and electropositive multimodal chromatography enhanced by advance chromatin extraction

A seamless and nonprotein A‐based monoclonal antibody purification platform using void‐exclusion anion exchange chromatography and electropositive multimodal chromatography enhanced by advance chromatin extraction

Study of catalase adsorption on two mixed‐mode ligands and the mechanism involved therein

Exploring the separation power of mixed‐modal resins for purification of recombinant osteopontin from clarified Escherichia coli lysates

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