High throughput screening based selection of phases for aqueous two-phase system-centrifugal partitioning chromatography of monoclonal antibodies

Oelmeier, Stefan A.; Effio, Christopher Ladd; Hubbuch, Jürgen

doi:10.1016/j.chroma.2012.06.075

Cited by 37 publications

(18 citation statements)

References 26 publications

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“…A high‐throughput screening procedure adapted from Ref. was used for protein precipitation studies. Purified water, salt stock solutions, and/or PEG solutions were pipetted into polypropylene 96‐well filter plates (Bio‐Inert®, Pall GmbH, Dreieich, DE) and mixed on the Te‐Shake ™ for 5 min at 1100 rpm.…”

Section: Methodsmentioning

confidence: 99%

“…In comparison with traditional methods, only fractional amounts of material was now required for the experiments. In the field of downstream processing, screening methods for chromatographic batch binding conditions, parallelized column chromatography in μL‐scale, solubility and precipitation screenings, and aqueous two‐phase extraction have been developed. The evolving demand for analytical techniques matching the speed of the experimentation was responded within the last years by both academia and industry.…”

Section: Introductionmentioning

confidence: 99%

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High‐throughput process development of purification alternatives for the protein avidin

Diederich

Hoffmann

Hubbuch

2015

Biotechnology Progress

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With an increased number of applications in the field of the avidin-biotin technology, the resulting demand for highly-purified protein avidin has drawn our attention to the purification process of avidin that naturally occurs in chicken egg white. The high-throughput process development (HTPD) methodology was exploited, in order to evaluate purification process alternatives to commonly used ion-exchange chromatography. In a high-throughput format, process parameters for aqueous two-phase extraction, selective precipitation with salts and polyethylene glycol, and hydrophobic interaction and mixed-mode column chromatography experiments were performed. The HTPD strategy was complemented by a high-throughput tandem high-performance liquid chromatography assay for protein quantification. Suitable conditions for the separation of avidin from the major impurities ovalbumin, ovomucoid, ovotransferrin, and lysozyme were identified in the screening experiments. By combination of polyethylene glycol precipitation with subsequent resolubilization and separation in a polyethylene glycol/sulfate/sodium chloride two-phase system an avidin purity of 77% was obtained with a yield >90% while at the same time achieving a significant reduction of the process volume. The two-phase extraction and precipitation results were largely confirmed in larger scale with scale-up factors of 230 and 133, respectively. Seamless processing of the avidin enriched bottom phase was found feasible by using mixed-mode chromatography. By gradient elution a final avidin purity of at least 97% and yield >90% was obtained in the elution pool. The presented identification of a new and beneficial alternative for the purification of the high value protein thus represents a successful implementation of HTPD for an industrially relevant purification task.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

High‐throughput process development of purification alternatives for the protein avidin

Diederich

Hoffmann

Hubbuch

2015

Biotechnology Progress

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“…S1 in the Supplementary material). Due to the shear forces between both phases, the mobile phase is dispersed in the stationary phase and thus, biochemical products using CPC have been reported in the last years [8][9][10][11][12][13]. Thereby, aqueous two-phase systems (ATPS) were preferred as systems.…”

Section: Introductionmentioning

confidence: 99%

Separation of active laccases from Pleurotus sapidus culture supernatant using aqueous two-phase systems in centrifugal partition chromatography

Schwienheer

Zeiner

Merz

2015

Journal of Chromatography B

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“…Because the partitioning of biomolecules is influenced by various factors, such as biomolecule hydrophobicity, charge, structure, size, as well as ABS features (PEG molecular weight, salt type, pH, additives), considering the lack of predictive models for ABS selection envisaging a specific purification problem, a lot of experiments for each ABS are necessary in order to gain sufficient insights . Given that, an automated approach in obtaining these insights is highly beneficial.…”

Section: Introductionmentioning

confidence: 99%

High‐throughput screening of aqueous biphasic systems with ionic liquids as additives for extraction and purification of enveloped virus‐like particles

Marchel

Soares

Vormittag

et al. 2019

Engineering Reports

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Due to their unique properties, virus‐like particles (VLPs) have been portrayed as a promising high‐value biopharmaceutical in VLP‐based vaccination and cancer therapy. Nevertheless, due to limited physical and economical capabilities of the current downstream processing of VLPs, their production is still difficult and seen as a major problem that needs to be tackled. In this work, high‐throughput screening on a liquid handling station (LHS) has been implemented for efficient selection of adequate polymer‐salt aqueous biphasic systems (ABS) for extraction and purification of enveloped Hepatitis C virus pseudoparticles (HCVpp). The effect of polyethylene glycol (PEG) molecular weight and salt type (citrate, sulfate, and phosphate) was first evaluated. Furthermore, to optimize extraction parameters, the effect of pH and tie‐line lengths (TLL) was also addressed. For the most promising ABS and extraction conditions, the addition of ten ionic liquids (ILs) as adjuvants was investigated. Insights on the chemical features of ILs that impact HCV‐VLPs partitioning are highlighted. Finally, the potential of studied ABS containing ILs as adjuvants in the extraction and purification of VLPs from cell culture supernatants was addressed. The 100% extraction efficiency of VLPs in the PEG‐rich phase was achieved, and VLP purity was increased compared to the system without IL. In conclusion, these promising results show that ILs can be very effective in modulating the phase properties of polymer‐salt ABS, achieving high HCV‐VLP purification.

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High throughput screening based selection of phases for aqueous two-phase system-centrifugal partitioning chromatography of monoclonal antibodies

Cited by 37 publications

References 26 publications

High‐throughput process development of purification alternatives for the protein avidin

High‐throughput process development of purification alternatives for the protein avidin

Separation of active laccases from Pleurotus sapidus culture supernatant using aqueous two-phase systems in centrifugal partition chromatography

High‐throughput screening of aqueous biphasic systems with ionic liquids as additives for extraction and purification of enveloped virus‐like particles

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