New Technology for High-Performance and High-Capacity Bio-separations: Protein Purification using "Hydrophobic Water" Solvent Systems
Lead Research Organisation:
Queen's University Belfast
Department Name: Sch of Chemistry and Chemical Eng
Abstract
New Technology for High-Performance and High-Capacity Bio-separations: Protein Purification using "Hydrophobic Water" Solvent Systems in Countercurrent Chromatography Apparatus
Summary
In the biotechnology industries, the manufacture of high-value proteins, peptides and biomolecules require fast, low cost, reliable, GMP-compliant and scalable production methodologies. One scientific area used to achieve this, is the use of preparatory chromatography and related extraction technologies. It is proposed to use a combination of three relatively new technologies, namely: (1) ionic liquids, (2) modern high performance countercurrent chromatography (HPCCC) and centrifugal partition chromatography (CPC) and (3) co-solvent free, high water content microemulsions derived from catanionic surfactants. The microemulsion solvent systems proposed are a class of surface active, hydrophobic ionic liquids, which enable a new range of high performance, high capacity and environmentally friendly separations of peptides, proteins and other biomolecules. The mobile or carrier phase in these separations is usually water, which makes the process cheap and environmentally benign. This is a highly multidisciplinary project, which will combine elements of bioscience, biochemistry, chemical engineering, physical, and synthetic chemistry. This project will make use of ionic liquids solvent systems and microemulsion solvent systems in the solvent extraction and countercurrent chromatographic preparation, purification and isolation of peptides and proteins of commercial importance. This will initially use model separations of proteins, to develop and integrate the different technological parts of this new methodology, and then use this information to carry out separations of proteins mixtures from natural sources. Also, this project aims to scale up the protein separations by the use of high capacity CPC equipment installed in the QUILL Research Centre and employ automation to reduce the running costs of the separations. The ultimate goal of this project is to produce a general purpose, clean, water based, efficient, cost effective peptide and protein separation system, that can be exploited by the UK biotechnology industry and help in the growth of these sectors in the UK economy.
Summary
In the biotechnology industries, the manufacture of high-value proteins, peptides and biomolecules require fast, low cost, reliable, GMP-compliant and scalable production methodologies. One scientific area used to achieve this, is the use of preparatory chromatography and related extraction technologies. It is proposed to use a combination of three relatively new technologies, namely: (1) ionic liquids, (2) modern high performance countercurrent chromatography (HPCCC) and centrifugal partition chromatography (CPC) and (3) co-solvent free, high water content microemulsions derived from catanionic surfactants. The microemulsion solvent systems proposed are a class of surface active, hydrophobic ionic liquids, which enable a new range of high performance, high capacity and environmentally friendly separations of peptides, proteins and other biomolecules. The mobile or carrier phase in these separations is usually water, which makes the process cheap and environmentally benign. This is a highly multidisciplinary project, which will combine elements of bioscience, biochemistry, chemical engineering, physical, and synthetic chemistry. This project will make use of ionic liquids solvent systems and microemulsion solvent systems in the solvent extraction and countercurrent chromatographic preparation, purification and isolation of peptides and proteins of commercial importance. This will initially use model separations of proteins, to develop and integrate the different technological parts of this new methodology, and then use this information to carry out separations of proteins mixtures from natural sources. Also, this project aims to scale up the protein separations by the use of high capacity CPC equipment installed in the QUILL Research Centre and employ automation to reduce the running costs of the separations. The ultimate goal of this project is to produce a general purpose, clean, water based, efficient, cost effective peptide and protein separation system, that can be exploited by the UK biotechnology industry and help in the growth of these sectors in the UK economy.
Technical Summary
New Technology for High-Performance and High-Capacity Bio-separations: Protein Purification using "Hydrophobic Water" Solvent Systems in Countercurrent Chromatography Apparatus
Technical Summary
This project makes use of newly discovered ionic liquids and microemulsion solvent systems derived from ionic liquids, for use in modern countercurrent chromatographic (CCC) equipment. CCC separations are based on the partitioning of solutes between two immiscible solvents. It is proposed that a combination of (1) ionic liquids, (2) modern high performance countercurrent chromatography (HPCCC) and centrifugal partition chromatography (CPC) and (3) co-solvent free, high water content microemulsions be used to separate peptides and proteins. The microemulsion solvent systems that would be used contain a surface active, hydrophobic ionic liquid / water / hexane mixture. These have water as a mobile phase and a high-water content microemulsion as a stationary phase in the CCC machine. This will enable a range of high performance, high capacity and environmentally friendly separations of peptides, proteins and other biomolecules to be carried out. Since the mobile or carrier phase in these separations is usually water, this makes the separation process cost effective, environmentally benign, robust and tolerant of suspended solid matter in the samples (which would normally destroy a HPLC column). In addition, the whole process can be scaled up and automated to minimize protein production costs.
Technical Summary
This project makes use of newly discovered ionic liquids and microemulsion solvent systems derived from ionic liquids, for use in modern countercurrent chromatographic (CCC) equipment. CCC separations are based on the partitioning of solutes between two immiscible solvents. It is proposed that a combination of (1) ionic liquids, (2) modern high performance countercurrent chromatography (HPCCC) and centrifugal partition chromatography (CPC) and (3) co-solvent free, high water content microemulsions be used to separate peptides and proteins. The microemulsion solvent systems that would be used contain a surface active, hydrophobic ionic liquid / water / hexane mixture. These have water as a mobile phase and a high-water content microemulsion as a stationary phase in the CCC machine. This will enable a range of high performance, high capacity and environmentally friendly separations of peptides, proteins and other biomolecules to be carried out. Since the mobile or carrier phase in these separations is usually water, this makes the separation process cost effective, environmentally benign, robust and tolerant of suspended solid matter in the samples (which would normally destroy a HPLC column). In addition, the whole process can be scaled up and automated to minimize protein production costs.
Planned Impact
The research will have a direct impact on biotechnology and pharmaceutical industries, and on society, contributing to the development of a new separation technology suitable for scale up for commercial production of peptides, proteins, bio-chemicals and pharmaceuticals.
Industry:
Ionic liquid-liquid chromatography (ILLC) and ionic liquid micellar chromatography (ILMC) are well suited to the cost effective, clean, sustainable extraction and purification of peptides, proteins and other bio-chemicals. This will enable the extraction of high value peptides and proteins from various sources. The ionic liquid-liquid extraction and chromatography technology (using a water mobile phase) also offers the possibility of expansion, from laboratory to industrial scale, which, given the full recyclability of ionic liquids and other solvents used, the low toxicity of the ionic liquids, and the benefits of a highly intensified water based technology, generates higher operational safety margins, and the potential benefits of reduced costs. This process can be fully automated through the use of computer controlled pumps and switching valves, and allow semi-continuous or continuous separations, which are preferred by industry.
The following activities will ensure effective communication of the project results to the relevant stakeholders and their engagement for further exploitation of the ILLC and ILMC technology.
Links with process industries in existing research and industrial consortia:
We have substantial links with process industries via QUILL, with an industrial consortium. The consortia will be used as an avenue to disseminate the results of the work, and to engage with industries with potential applications of ILLC and ILMC.
Presentations in conferences and publications:
The outcomes of this innovative technology will be disseminated through QUILL via patenting and licensing intellectual property (through our partners Mathys and Squire) and publications in high impact journals.
Society
Many current methods of producing peptides and proteins generally work on small scale. Currently there is a demand research and medicinal products based on peptides and proteins, and ILLC and ILMC will provide a direct means for extracting and purifying such compounds, via a sustainable, environmentally friendly, easily scalable and automated process. The technology we aim to produce can generate significant quantities of isolated therapeutic compounds, extending its benefits to the wide community.
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We plan to disseminate the results of the project to the general public through activities such as articles in general publications, articles that discuss the importance of intensified separation technologies, of ionic liquids and of their application and producing therapeutic compounds. This will be posted on the Universities web sites and in various publications.
Industry:
Ionic liquid-liquid chromatography (ILLC) and ionic liquid micellar chromatography (ILMC) are well suited to the cost effective, clean, sustainable extraction and purification of peptides, proteins and other bio-chemicals. This will enable the extraction of high value peptides and proteins from various sources. The ionic liquid-liquid extraction and chromatography technology (using a water mobile phase) also offers the possibility of expansion, from laboratory to industrial scale, which, given the full recyclability of ionic liquids and other solvents used, the low toxicity of the ionic liquids, and the benefits of a highly intensified water based technology, generates higher operational safety margins, and the potential benefits of reduced costs. This process can be fully automated through the use of computer controlled pumps and switching valves, and allow semi-continuous or continuous separations, which are preferred by industry.
The following activities will ensure effective communication of the project results to the relevant stakeholders and their engagement for further exploitation of the ILLC and ILMC technology.
Links with process industries in existing research and industrial consortia:
We have substantial links with process industries via QUILL, with an industrial consortium. The consortia will be used as an avenue to disseminate the results of the work, and to engage with industries with potential applications of ILLC and ILMC.
Presentations in conferences and publications:
The outcomes of this innovative technology will be disseminated through QUILL via patenting and licensing intellectual property (through our partners Mathys and Squire) and publications in high impact journals.
Society
Many current methods of producing peptides and proteins generally work on small scale. Currently there is a demand research and medicinal products based on peptides and proteins, and ILLC and ILMC will provide a direct means for extracting and purifying such compounds, via a sustainable, environmentally friendly, easily scalable and automated process. The technology we aim to produce can generate significant quantities of isolated therapeutic compounds, extending its benefits to the wide community.
.
We plan to disseminate the results of the project to the general public through activities such as articles in general publications, articles that discuss the importance of intensified separation technologies, of ionic liquids and of their application and producing therapeutic compounds. This will be posted on the Universities web sites and in various publications.
| Description | The importance and motivation for this research is that ionic liquids have been found to be excellent solvents for proteins and have been demonstrated to work in a highly effective manner in counter-current chromatography (CCC). Taken together, this enables peptide and protein separations to be carried out at much higher concentrations and space time yields, using relatively small amounts of solvent, than is currently possible with conventionally used solvent systems. Since CCC separations with ionic liquids and high water content microemulsions behave in a different manner, and have different selectivities, when compared to HPLC, gel or size exclusion chromatography, electrophoresis and other methodologies used in protein purification, it will complement currently available technologies. This research is based on the discovery of a new "revolutionary" triphasic solvent system and new biphasic water / hydrophobic microemulsion solvent systems based on ethyl ethanoate. These separations use the cheapest liquid solvent available (water) as a mobile phase and relatively cheap organic solvents such as hexane or ethyl ethanoate. The ionic liquid which has been tested is at most 1.5 mol% or 10 wt% of the stationary phase making separations economical when compares to aqueous biphasic solvent systems based of K2[HPO4] / PEG or K2[HPO4] / [C4mim]Cl. These separations are generally tolerant of solids which would normally block or destroy a HPLC or gel chromatography column, making the CPC separations much more robust and insensitive to particulate matter often found in crude protein samples. The water / ionic liquid + organic solvent microemulsion solvent systems used in CPC separations have solute capacities of 1 to 2 order of magnitude higher than those used in HPLC, and since the stationary phase is a liquid, rather than a surface (as in solid HPLC columns), the stationary phase can have much higher solute capacities than is found in similarly sized HPLC columns. Thus, far lower quantities of solvent per gram of protein are required in CPC separations, than is the case for HPLC separations. These ionic liquid and microemulsion solvent systems, combined with automated CPC apparatus and technologies have the potential to revolutionise the way in which proteins, enzymes and biopolymers are produced and dramatically reduce the production costs. A model protein separation of lysozyme / cytochrome C was tested to determine whether microemulsion solvent systems can be used in separations using CPC instrument. The solvent system chosen was a water / ethyl ethanoate solvent system containing the ionic liquid [C10mim][DiIOP]. The distribution ratio of lysozyme between water and ethyl ethanoate containing [C10mim][DiIOP] was measured by UV-Vis spectroscopy. A biphasic solution containing water and ethyl ethanoate was prepared and 10 cm3 of each phase of this solvent system were placed in a 40 cm3 centrifuge tube. 25 mg of lysozyme was added and the mixture shaken until the lysozyme dissolved. The distribution ratio of lysozyme was then determined by dividing the absorbance of lysozyme in the stationary (ethyl ethanoate) phase by the absorption of lysozyme dissolved in the aqueous mobile phase. Increasing amounts of [C10mim][DiIOP] were added to the mixture in the centrifuge tube, and the absorptions at 280 nm were determined for both phases, and the variation of distribution ratio was calculated. We could demonstrate that the ionic liquid [C10mim][DiIOP] causes water to dissolve in the hydrophobic ethyl ethanoate phase, and generates an aqueous biphasic solvent system, the composition of which, and the water content of the stationary phase can be controlled by the amount of ionic liquid added. The aqueous MP composition remains relatively stable, remaining at 1.5 to 2.5 mol % ethyl ethanoate and 98.5-97.5 mol% water, as the amount of ionic liquid [C10mim][DiIOP] is increased in the solvent system. From these data, the distribution ratio of the ionic liquid remains at 40-50. It is now possible to generate aqueous biphasic solvent systems which have a highly variable water content in the water immiscible stationary phase. As a result, solvent systems can be designed to suit a particular protein separation process. |
| Exploitation Route | A manuscript of a publication has been submitted to the journal "Green Chemistry" and is currently under review. Furthermore, there have been several requests from different industries for specific protein separations, which are currently tested. |
| Sectors | Agriculture, Food and Drink,Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology |
| Description | It is now possible to generate aqueous biphasic solvent systems which have a highly variable water content in the water immiscible stationary phase. As a result, solvent systems can be designed to suit a particular protein separation process. This has received interest from industrial partners to test the method on lentinan separation and purification from mushrooms; the testing for a local industrial partner has been continued and is still in progress. |
| First Year Of Impact | 2019 |
| Sector | Pharmaceuticals and Medical Biotechnology |
| Description | Follow-up collaboration for scale-up processing and separation of lentinan with Hughes Mushrooms |
| Organisation | Hughes Group |
| Country | United Kingdom |
| Sector | Private |
| PI Contribution | The work on CCC separation with ionic liquids has led to a partnership with Hughes Mushrooms, who borrowed the CCC equipment to scale up lentinan production for 6 months, which was carried out between March 2020 - September 2020. The researcher involved in the project, Martyn Earle, had been employed as a consultant for the industrial partner by Hughes Mushrooms providing technical insights and practical advice on the separation technology. |
| Collaborator Contribution | Hughes Mushrooms (https://hughesmushrooms.com) borrowed the CCC equipment from the QUILL Research Centre to scale up lentinan production for 6 months, which was carried out between March 2020 - September 2020. The company are exploring further commercialisation of the product and the feasibility of the separation method. |
| Impact | Currently there is no direct output, however, the commercialisation of the separation process for lentinan is being further explored. |
| Start Year | 2020 |