Final Report Summary - MEMTIDE (Membrane Enhanced Tide Synthesis - A New Paradigm Peptide / Oligonucleotide Synthesis Technology)
1. INTRODUCTION
The MemTide project was initiated in December 2009 with the objective of creating new paradigms for tide synthesis by coupling emerging membrane technology with state of the art peptide and oligonucleotide synthesis techniques. MemTide involved 6 partners from 5 different European countries (3 academic institutes, 1 SME with a linked enterprise, 1 fine chemicals company, 1 pharmaceuticals manufacturer). A total of 10 Early Stage Researchers and 5 Experienced Researchers were involved in the project, which trained the young researchers with both a broad set of professional skills and valuable expertise in emerging membrane and tide synthesis technologies
2. RESULTS ACHIEVED
Polyimide membranes were manufactured at an industrial scale and rolled into spiral-wound type modules of different sizes. It was found that the membrane and modules are stable in many organic solvents. Subsequently, spiral wound modules of 1.8” diameter containing commercially available DuraMem 300 membranes were made and tested with consistent performance achieved over a two month period and no stability issues observed.
Crosslinking was used with polyimide OSN membranes, and these membranes were shown to be stable in all ranges of water-acetonitrile mixture. Further experiments showed that DBX-crossilnked PBI membranes are stable in aggressive solvents such as DMF as well as polar solvents such as MeOH and acetonitrile.
A standard peptide was synthesised on solid phase and a soluble polymeric support was designed and synthesis. These polymers were performed in order to anchor the standard peptide on them. In order to accomplish the goals of the project, it was necessary to design a methodology to obtain longer peptide, after the standard peptide anchored on a soluble polymeric support has been synthesized.
Peptide synthesis was performed using different coupling agents and solvents, and was carried out under different conditions in order to optimise the reaction. To improve results, IRB used ‘Click Chemistry’ to carry out the coupling between polymers and the standard peptide fragment. High rejection rates were observed with commercially available membranes. Ultimately, the synthesis of a commercial four-amino-acid peptide with OSN as isolation technique was successful. High yield and purity were achieved with close to equivalent amino acids used in couplings.
Soluble supports and linkers were developed for use in oligonucleotide synthesis. The rejections of these compounds against available membranes showed encouraging results. A two-stage solvent recovery process was established and tested: this process will allow purification of oligonucleotide and peptide mixtures without significant volume of solvents. A new platform called Membrane enhanced oligonucleotide synthesis (MEOS) was successfully demonstrated by synthesizing up to pentaribonucleotides on soluble PEG support. Using new PBI membranes, a complete separation of excess reagents from growing nucleotide was achieved. 5-mer DNA oligonucleotides were prepared in solution on a soluble support using both phosphoramidite chemistry and phosphotriester chemistry. The purity obtained was comparable with or even superior to that on solid support.
3. CONCLUSIONS
Integrally skinned asymmetric polybenzimidazole (PBI) nanofiltration membranes were successfully prepared and crosslinked using halogenated compounds – dibromoxylene (DBX) and dibromobutane (DBB). Both membranes showed consistent and reliable batch to batch separation performance in acetontrile, DMF and DI water using a range of PEGs as nanofiltration markers. In addition, DBX crosslinked PBI membranes exhibit permeabilities superior to those of DBB crosslinked ones in all tested solvents.
In the area of peptide synthesis, it was determined that OH-BTL-resin was more acid-stable and more efficient for SPPS than Wang resin. Using this new resin, three model peptides were obtained with higher yields and purities when compared with those achieved with Wang resin. Different commercial soluble supports were assayed in LPPS, with MeO-PEG-NH2 showing the best results. The OSN assisted LPPS of model peptide: H-RADA-NH2 was successfully carried out on the support PyPEG. The scale-up of the three novel soluble supports synthesis were carried out. Taking into account reaction conditions, safety and costs, PyPEG was found to be the best choice.
It was also shown that the new process (LPPS with OSN) can produce the model peptide (Fmoc-RADA) with high yield and purity (82.0 and 98.5% respectively before cleavage and global deprotection). As compared with SPPS, in which at least 1.50 eq amino acids are needed for couplings, close-to-equivalent amino acids are sufficient to drive all the couplings to completion with short reaction time (30 minutes).
A new liquid-phase oligonucleotide synthesis (LPOS) platform was introduced, using organic solvent nanofiltration (OSN) as a scalable purification technology. By growing oligonucleotides on a soluble and branched monodispersed PEG support, it was determined that purification after each reaction can be performed using OSN membranes exploiting the difference in size. Although we have shown the proof-of-principle for a potentially new paradigm for oligos synthesis, a continual improvements and optimization steps must be taken. Several process challenges have been identified and possible solutions have been suggested. The proposed LPOS-OSN platform presents an interesting alternative for large-scale synthesis of oligos and it is expected that synergistic combination of LPS and SPS technology will allow economical synthesis of oligo-based therapeutics required in metric-ton scale.
The concept of synthesis of fully protected oligotrimers on a a soluble tetrakis-O-(4-azidomethylphenyl)pentaerythritol support bearing four thymidines attached through “Q-linker” proved to be successful.
4. EXPECTED FINAL RESULTS AND IMPACT
The ultimate objective for MemTide is to make a paradigm shift in the technology used for the synthesis of tides by coupling the project’s innovations in OSN and tide synthesis to devise a breakthrough technology – membrane enhanced tide synthesis. Membrane separation between reaction cycles during solution phase synthesis offers major advantages over solid phase synthesis. The advantages of “classical” solution phase synthesis are combined with the elegant separation of the solid phase method. Reaction in the solution phase provides a faster reaction rate, is less affected by steric hindrance due to folding or reactions within confined space (for example transpeptidation), and is not limited by intraparticle diffusional mass transfer phenomena that adversely affect solid phase synthesis.
Results stemming from MemTide research are likely to have a number of applications, as described below:
Newly developed PBI membranes show similar performance to polyimide membranes, but can withstand much wider chemical environments. Hence, they have great potential to be used as OSN membranes in various pharmaceutical purification processes which also employ acids and bases. In addition, PBI seems to be a promising membrane material for the needs of CCS.
The main advantage of the new LPOS process is that its performance is not affected by the scale of synthesis. Hence, for instance, a convergent synthesis protocol by coupling trimers together using LPOS platform, where the trimers are prepared via SPOS technology, would be a feasible option to investigate further. With ever-growing number of oligo-based drugs going through clinical trials, a scalable manufacturing technology is expected to further boost industry investment in this exciting new era of oligo therapeutics. Similarly, in the peptide field, the business case study suggests that that LPPS with OSN has great potential for more cost-effective peptide synthesis process.
During the project we have developed novel Cys protecting groups and new disulfide formation strategies. The S-Tmp protecting group was commercialized by Nova Biochem and S-Dmp by IRIS biotech.
Moving forward, the development of the previous protocols will be employed for the synthesis of oligonucleotides using a polyethyleneglycol (PEG) soluble support and the alternative acetal protection using RNA building blocks. This work will be a collaboration between the University of Turku (which has expertise oligonucleotide chemistry) and the Imperial College of London, (which has OSN expertise) system. The procedure will be simplified in a two-step reaction protocol. This collaborative work between the Imperial College of London and the University of Turku will try to demonstrate that we can achieve 100% deprotection by using the alternative acetal protection, as it is much smaller than the DMTr group.
Results have been disseminated widely through a variety of means. 11 papers resulting from MemTide’s research have been published thus far, with 1 more paper accepted for publication and a further 9 currently in preparation. MemTide’s researchers have in total given 11 oral and 16 poster presentations at 15 international conferences.
Further results with commercial potential will be protected through patent filing, which will in turn become public record as the applications are published in national and European patent journals. The project’s work will continue to be relevant after the lifespan of MemTide has ended, with aspects being introduced into training and lecture courses at the universities involved, and publications based on MemTide work continuing to be produced. ESRs and ERs will move into new organisations, taking their knowledge of techniques and best practices with them. Their knowledge of new tide manufacturing technology will be valuable not only in their own careers, but also in wider society as MemTide’s researchers bring their work to bear on the development of new therapies and technologies.
5. CONTACT DETAILS
Professor Andrew Livingston
MemTide Project Coordinator
Department of Chemical Engineering
Imperial College London
South Kensington Campus
London SW7 2AZ
a.livingston@imperial.ac.uk
6. PROJECT WEBSITE
http://www.imperial.ac.uk/memtide
The MemTide project was initiated in December 2009 with the objective of creating new paradigms for tide synthesis by coupling emerging membrane technology with state of the art peptide and oligonucleotide synthesis techniques. MemTide involved 6 partners from 5 different European countries (3 academic institutes, 1 SME with a linked enterprise, 1 fine chemicals company, 1 pharmaceuticals manufacturer). A total of 10 Early Stage Researchers and 5 Experienced Researchers were involved in the project, which trained the young researchers with both a broad set of professional skills and valuable expertise in emerging membrane and tide synthesis technologies
2. RESULTS ACHIEVED
Polyimide membranes were manufactured at an industrial scale and rolled into spiral-wound type modules of different sizes. It was found that the membrane and modules are stable in many organic solvents. Subsequently, spiral wound modules of 1.8” diameter containing commercially available DuraMem 300 membranes were made and tested with consistent performance achieved over a two month period and no stability issues observed.
Crosslinking was used with polyimide OSN membranes, and these membranes were shown to be stable in all ranges of water-acetonitrile mixture. Further experiments showed that DBX-crossilnked PBI membranes are stable in aggressive solvents such as DMF as well as polar solvents such as MeOH and acetonitrile.
A standard peptide was synthesised on solid phase and a soluble polymeric support was designed and synthesis. These polymers were performed in order to anchor the standard peptide on them. In order to accomplish the goals of the project, it was necessary to design a methodology to obtain longer peptide, after the standard peptide anchored on a soluble polymeric support has been synthesized.
Peptide synthesis was performed using different coupling agents and solvents, and was carried out under different conditions in order to optimise the reaction. To improve results, IRB used ‘Click Chemistry’ to carry out the coupling between polymers and the standard peptide fragment. High rejection rates were observed with commercially available membranes. Ultimately, the synthesis of a commercial four-amino-acid peptide with OSN as isolation technique was successful. High yield and purity were achieved with close to equivalent amino acids used in couplings.
Soluble supports and linkers were developed for use in oligonucleotide synthesis. The rejections of these compounds against available membranes showed encouraging results. A two-stage solvent recovery process was established and tested: this process will allow purification of oligonucleotide and peptide mixtures without significant volume of solvents. A new platform called Membrane enhanced oligonucleotide synthesis (MEOS) was successfully demonstrated by synthesizing up to pentaribonucleotides on soluble PEG support. Using new PBI membranes, a complete separation of excess reagents from growing nucleotide was achieved. 5-mer DNA oligonucleotides were prepared in solution on a soluble support using both phosphoramidite chemistry and phosphotriester chemistry. The purity obtained was comparable with or even superior to that on solid support.
3. CONCLUSIONS
Integrally skinned asymmetric polybenzimidazole (PBI) nanofiltration membranes were successfully prepared and crosslinked using halogenated compounds – dibromoxylene (DBX) and dibromobutane (DBB). Both membranes showed consistent and reliable batch to batch separation performance in acetontrile, DMF and DI water using a range of PEGs as nanofiltration markers. In addition, DBX crosslinked PBI membranes exhibit permeabilities superior to those of DBB crosslinked ones in all tested solvents.
In the area of peptide synthesis, it was determined that OH-BTL-resin was more acid-stable and more efficient for SPPS than Wang resin. Using this new resin, three model peptides were obtained with higher yields and purities when compared with those achieved with Wang resin. Different commercial soluble supports were assayed in LPPS, with MeO-PEG-NH2 showing the best results. The OSN assisted LPPS of model peptide: H-RADA-NH2 was successfully carried out on the support PyPEG. The scale-up of the three novel soluble supports synthesis were carried out. Taking into account reaction conditions, safety and costs, PyPEG was found to be the best choice.
It was also shown that the new process (LPPS with OSN) can produce the model peptide (Fmoc-RADA) with high yield and purity (82.0 and 98.5% respectively before cleavage and global deprotection). As compared with SPPS, in which at least 1.50 eq amino acids are needed for couplings, close-to-equivalent amino acids are sufficient to drive all the couplings to completion with short reaction time (30 minutes).
A new liquid-phase oligonucleotide synthesis (LPOS) platform was introduced, using organic solvent nanofiltration (OSN) as a scalable purification technology. By growing oligonucleotides on a soluble and branched monodispersed PEG support, it was determined that purification after each reaction can be performed using OSN membranes exploiting the difference in size. Although we have shown the proof-of-principle for a potentially new paradigm for oligos synthesis, a continual improvements and optimization steps must be taken. Several process challenges have been identified and possible solutions have been suggested. The proposed LPOS-OSN platform presents an interesting alternative for large-scale synthesis of oligos and it is expected that synergistic combination of LPS and SPS technology will allow economical synthesis of oligo-based therapeutics required in metric-ton scale.
The concept of synthesis of fully protected oligotrimers on a a soluble tetrakis-O-(4-azidomethylphenyl)pentaerythritol support bearing four thymidines attached through “Q-linker” proved to be successful.
4. EXPECTED FINAL RESULTS AND IMPACT
The ultimate objective for MemTide is to make a paradigm shift in the technology used for the synthesis of tides by coupling the project’s innovations in OSN and tide synthesis to devise a breakthrough technology – membrane enhanced tide synthesis. Membrane separation between reaction cycles during solution phase synthesis offers major advantages over solid phase synthesis. The advantages of “classical” solution phase synthesis are combined with the elegant separation of the solid phase method. Reaction in the solution phase provides a faster reaction rate, is less affected by steric hindrance due to folding or reactions within confined space (for example transpeptidation), and is not limited by intraparticle diffusional mass transfer phenomena that adversely affect solid phase synthesis.
Results stemming from MemTide research are likely to have a number of applications, as described below:
Newly developed PBI membranes show similar performance to polyimide membranes, but can withstand much wider chemical environments. Hence, they have great potential to be used as OSN membranes in various pharmaceutical purification processes which also employ acids and bases. In addition, PBI seems to be a promising membrane material for the needs of CCS.
The main advantage of the new LPOS process is that its performance is not affected by the scale of synthesis. Hence, for instance, a convergent synthesis protocol by coupling trimers together using LPOS platform, where the trimers are prepared via SPOS technology, would be a feasible option to investigate further. With ever-growing number of oligo-based drugs going through clinical trials, a scalable manufacturing technology is expected to further boost industry investment in this exciting new era of oligo therapeutics. Similarly, in the peptide field, the business case study suggests that that LPPS with OSN has great potential for more cost-effective peptide synthesis process.
During the project we have developed novel Cys protecting groups and new disulfide formation strategies. The S-Tmp protecting group was commercialized by Nova Biochem and S-Dmp by IRIS biotech.
Moving forward, the development of the previous protocols will be employed for the synthesis of oligonucleotides using a polyethyleneglycol (PEG) soluble support and the alternative acetal protection using RNA building blocks. This work will be a collaboration between the University of Turku (which has expertise oligonucleotide chemistry) and the Imperial College of London, (which has OSN expertise) system. The procedure will be simplified in a two-step reaction protocol. This collaborative work between the Imperial College of London and the University of Turku will try to demonstrate that we can achieve 100% deprotection by using the alternative acetal protection, as it is much smaller than the DMTr group.
Results have been disseminated widely through a variety of means. 11 papers resulting from MemTide’s research have been published thus far, with 1 more paper accepted for publication and a further 9 currently in preparation. MemTide’s researchers have in total given 11 oral and 16 poster presentations at 15 international conferences.
Further results with commercial potential will be protected through patent filing, which will in turn become public record as the applications are published in national and European patent journals. The project’s work will continue to be relevant after the lifespan of MemTide has ended, with aspects being introduced into training and lecture courses at the universities involved, and publications based on MemTide work continuing to be produced. ESRs and ERs will move into new organisations, taking their knowledge of techniques and best practices with them. Their knowledge of new tide manufacturing technology will be valuable not only in their own careers, but also in wider society as MemTide’s researchers bring their work to bear on the development of new therapies and technologies.
5. CONTACT DETAILS
Professor Andrew Livingston
MemTide Project Coordinator
Department of Chemical Engineering
Imperial College London
South Kensington Campus
London SW7 2AZ
a.livingston@imperial.ac.uk
6. PROJECT WEBSITE
http://www.imperial.ac.uk/memtide