Periodic Reporting for period 1 - RandoPEGMed (Random Copolymers Enabling Nonimmunogenic PEGylation for Medical Therapeutics)
Reporting period: 2022-10-01 to 2025-03-31
PEGylation, the attachment of the highly hydrophilic, biocompatible polymer polyethylene glycol (PEG) - common in many cosmetic and pharmaceutical products used every day to therapeutics, has become a key strategy in current nanomedicine. PEGylation is indispensable for a broad variety of commercial therapeutics. They range from protein drugs to relevant nanocarriers, such as liposomes and lipid nanoparticles (LNP). PEG leads to the particular “stealth effect”, i.e. strongly prolonged blood circulation times and apparent “invisibility” for the immune system. This is also used for lipid nano-particles (LNP) transporting mRNA vaccines, used worldwide in recent years against the COVID-19 pandemic. However, this powerful weapon is now in danger of becoming blunt: An increasing body of literature from immunologists, clinicians and biochemists shows a rapid increase of individuals (50-70% in industrialized countries) possessing anti-PEG antibodies (APAs). This causes undesirable rapid elimination of PEGylated drugs from the bloodstream, hypersensitivity and strong allergic reactions. The project RandoPEGMed introduces PEG-derived random copolymers for nanomedicine. These PEG copolymers with well-defined chain length possess randomly distributed “point mutations” along the chains, which both disables recognition by the immune system and the formation of antibodies. We systematically tested the effect of a varied concentration of “synthetic point mutations” (0 to 74 mol% GME) in the polymer chains on the binding capability of backbone- and end group-specific APAs via competitive ELISA. The concentration-dependent interaction between the antibodies and the polyether is detected by a decrease of fluorescence intensity. A major decrease of the recognition by anti-PEG antibodies was observed with increasing GME content, which translates to diminished antigenicity. A broad range of the modified PEG-structures, called "rPEGs" has been studied, showing that the interaction with anti-PEG antibodies can be eliminated. In several cell-line studies (murine cells and human cells) it has been demonstrated that these random PEG copolymers retain all favorable properties of PEG, such as high aqueous solubility and hydration as well as excellent cell viability. These findings open up a variety of new options for "rPEGs" in nanomedicine, particularly for improved nanocarriers and may help to avoid allergic reactions in patients in the future. Currently, collaboration with industry aims at making rPEG lipids available commercially.
The focus was placed on copolymers of EO with GME (glycidyl methyl ether). As GME is a formal dimer of EO (C2nH4nOn; n = 1 (EO), x2 (GME)), all copolymers of EO and GME represent constitutional isomers of the highly established poly(ethylene glycol), PEG. This is a major advantage with respect to pharmaceutical and medical application of these materials, e.g. when targeting approval by regulatory agencies.
Key results:
- Scale up of ultrapure GME monomer synthesis to 200g scale by a skilled lab technician.
- Copolymerization of EO and GME (“rPEGs”): This key route of the project has been investigated in detail with respect to different content of GME in the polyether copolymers. The respective polyethers can now be prepared in all compositions and with low dispersity (Mw/Mn < 1.07) in pharma-grade quality. The choice of solvent is a crucial parameter.
- A descriptive terminology has been developed to specify both composition and degree of polymerization (DP) of the rPEGs.
Solubility and chain flexibility of rPEG copolymers in aqueous solution and related LCST of the polymers have been studied in great detail, relying on 3 key methods: (i) conventional turbidimetry; (ii) DOSY NMR spectroscopy and (iii) ESR spectroscopy using TEMPO-based spin probes. We studied the effect of a varied concentration of “synthetic point mutations” (0 to 74 mol% GME) on the binding capability of backbone- and end group-specific APAs via competitive ELISA. The concentration-dependent interaction between the APA and polyether is detected by a decrease of fluorescence intensity. With increasing GME content the antigenicity decreases strongly. With incorporation of 21 –74 mol%GME in the polyether, the affinity drops to only 0.4 – 4.6∙10-6% relative to mPEG117. Larger alkyl groups, such as ethyl, further reduce antibody interaction (ELISA). Series of well-defined rPEG lipids (in perfect structural analogy to DMG-PEG) have been prepared and compared to PEG lipids used in commercial LNP formulations. Close collaboration with the company Evonik has been established, aiming at market introduction of these novel, non-immunogenic lipids in early 2026.
Most important achievements of the project since the start in 10/2022:
1. Synthesis of well-defined rPEGs up to 30.000 g/mol with precisely controlled molecular weights (MALDI-TOF), dispersity < 1.12 (most samples < 1.06) and pharma-grade quality.
2. ABA triblock copolymers with disordered and flexible rPEG midblock and polylactide (PLLA) end blocks. The triblock copolymers are used to generate both non-immunogenic hydrogels and novel types of toughening agents for PLLA to fabricate strong and tough or very soft PLLA materials for medical implants.
3. Detailed understanding of random and gradient structures (and the underlying formation kinetics) obtained in different solvents in the EO/GME statistical copolymerization. Copolymerization in DMSO leads to fully random copolymers, while slight gradient structures are observed in toluene and anisole.
4. First synthesis and testing of rPEG lipids with high structural definition (SEC, MALDI-TOF, NMR characterization). Replacement of PEG by rPEG chains in the DMG lipid structure was achieved, and the rPEG-lipids have been incorporated in LNPs, showing good control of their size in the 70 – 100nm range. The rPEG lipids do not bind to anti-PEG antibodies (ELISA).
5. An example of bioconjugation with rPEG has recently been established using the highly immunogenic therapeutic protein uricase.
Key results:
- Scale up of ultrapure GME monomer synthesis to 200g scale by a skilled lab technician.
- Copolymerization of EO and GME (“rPEGs”): This key route of the project has been investigated in detail with respect to different content of GME in the polyether copolymers. The respective polyethers can now be prepared in all compositions and with low dispersity (Mw/Mn < 1.07) in pharma-grade quality. The choice of solvent is a crucial parameter.
- A descriptive terminology has been developed to specify both composition and degree of polymerization (DP) of the rPEGs.
Solubility and chain flexibility of rPEG copolymers in aqueous solution and related LCST of the polymers have been studied in great detail, relying on 3 key methods: (i) conventional turbidimetry; (ii) DOSY NMR spectroscopy and (iii) ESR spectroscopy using TEMPO-based spin probes. We studied the effect of a varied concentration of “synthetic point mutations” (0 to 74 mol% GME) on the binding capability of backbone- and end group-specific APAs via competitive ELISA. The concentration-dependent interaction between the APA and polyether is detected by a decrease of fluorescence intensity. With increasing GME content the antigenicity decreases strongly. With incorporation of 21 –74 mol%GME in the polyether, the affinity drops to only 0.4 – 4.6∙10-6% relative to mPEG117. Larger alkyl groups, such as ethyl, further reduce antibody interaction (ELISA). Series of well-defined rPEG lipids (in perfect structural analogy to DMG-PEG) have been prepared and compared to PEG lipids used in commercial LNP formulations. Close collaboration with the company Evonik has been established, aiming at market introduction of these novel, non-immunogenic lipids in early 2026.
Most important achievements of the project since the start in 10/2022:
1. Synthesis of well-defined rPEGs up to 30.000 g/mol with precisely controlled molecular weights (MALDI-TOF), dispersity < 1.12 (most samples < 1.06) and pharma-grade quality.
2. ABA triblock copolymers with disordered and flexible rPEG midblock and polylactide (PLLA) end blocks. The triblock copolymers are used to generate both non-immunogenic hydrogels and novel types of toughening agents for PLLA to fabricate strong and tough or very soft PLLA materials for medical implants.
3. Detailed understanding of random and gradient structures (and the underlying formation kinetics) obtained in different solvents in the EO/GME statistical copolymerization. Copolymerization in DMSO leads to fully random copolymers, while slight gradient structures are observed in toluene and anisole.
4. First synthesis and testing of rPEG lipids with high structural definition (SEC, MALDI-TOF, NMR characterization). Replacement of PEG by rPEG chains in the DMG lipid structure was achieved, and the rPEG-lipids have been incorporated in LNPs, showing good control of their size in the 70 – 100nm range. The rPEG lipids do not bind to anti-PEG antibodies (ELISA).
5. An example of bioconjugation with rPEG has recently been established using the highly immunogenic therapeutic protein uricase.
Synthesis of well-defined rPEGs up to 30.000 g/mol with precisely controlled molecular weights (MALDI-TOF), dispersity < 1.12 (most samples < 1.06) i.e. pharma-grade quality.
Synthesis and detailed testing of rPEG based lipids in collaboration with the German company Evonik represents an important breakthrough. Replacement of PEG by rPEG in the DMG lipid structure was achieved, and the respective rPEG-lipids have been incorporated in LNPs prepared by common, commercially employed microflow techniques. The results evidence that rPEG lipids do not alter or disable LNP formation. The rPEG lipids do not bind to anti-PEG antibodies (APAs), which was also demonstrated by detailed competitive ELISA studies.
Another breakthrough lies in the observed miscibility of rPEG with polylactide (PLLA) in the melt, which offers a perspective on plasticizing of “Resomer” type PLLA structures with the noncrystalline and highly biocompatible rPEGs. Demixing of blends of rPEG and PLLA is solely driven by crystallization of PLLA in such materials. We foresee a major impact for therapeutics based on these systems as well as potential for translation.
All developments mentioned here have enormous potential for societal impact, since they open novel options for nanomedicine as well as controlled-release systems and for degradable medical polymers based on PLLA and PLGA.
Synthesis and detailed testing of rPEG based lipids in collaboration with the German company Evonik represents an important breakthrough. Replacement of PEG by rPEG in the DMG lipid structure was achieved, and the respective rPEG-lipids have been incorporated in LNPs prepared by common, commercially employed microflow techniques. The results evidence that rPEG lipids do not alter or disable LNP formation. The rPEG lipids do not bind to anti-PEG antibodies (APAs), which was also demonstrated by detailed competitive ELISA studies.
Another breakthrough lies in the observed miscibility of rPEG with polylactide (PLLA) in the melt, which offers a perspective on plasticizing of “Resomer” type PLLA structures with the noncrystalline and highly biocompatible rPEGs. Demixing of blends of rPEG and PLLA is solely driven by crystallization of PLLA in such materials. We foresee a major impact for therapeutics based on these systems as well as potential for translation.
All developments mentioned here have enormous potential for societal impact, since they open novel options for nanomedicine as well as controlled-release systems and for degradable medical polymers based on PLLA and PLGA.