Executive Summary
This year's TIDES Europe welcomed professionals from around the world for live and on-demand presentations, keynotes, posters and panel discussions in a 100% virtual setting.
In this executive summary, biotech researcher and writer Dr Catarina Carrao looks back on the hottest sessions which explored the conference's key themes.
For Dr. Barbara Adamczyk, Senior Analytical Scientist of Advanced Drug Delivery at AstraZeneca, ASOs face several analytical challenges for approval into the clinic. For example, Liquid Chromatography (LC) is no longer sufficient for the deep impurity analysis demanded by the regulatory agencies.
A solution to this problem is to use Ion-Pair Reversed Liquid Chromatography coupled with Ultra-Violet Mass Spectrometry IP-LC-UV-MS (IP-LC-UV-MS), which is able to give identity, assay and purity/impurities information needed for oligonucleotide Quality Control (QC).
Dr. Adamczyk clarified that Ionis Pharmaceuticals partners with AstraZeneca in regard to IP-LC-UV-MS oligonucleotide analysis. This method allows to quantify over 30 types of impurities in different reporting classes.
But, to Dr. Adamczyk, there are also opportunities to explore. For example, when transfer of complex methods is not feasible, there is an opportunity to use UV260 for oligo assay determination in-process control, which is easy to transfer and doesn’t require a working-solution standard.
The release testing is then LC-MS, which is performed internally at AstraZeneca in this case, with an assay acceptance criterion of 90-110%. Also, in-house, are the safety assessment studies performed, with a full-package for oligonucleotide clinical candidates and a limited package for oligonucleotide surrogates, which is essential in order to minimize off-target effects.
In relation to the question of performing only routine QC analysis, or get deeper insights into the oligonucleotides, Dr. Adamczyk emphasized the importance of collaboration between AstraZeneca and academies (e.g., Prof. Torgny Fornstedt laboratory at Karlstad University).
The collaborations allow an increase of knowledge regarding oligonucleotide separation mechanisms; possible alternatives for purity analysis of therapeutic oligos; and, to develop theoretical models and computer simulation algorithms that can correctly account for the complex behaviour of large molecules.
In fact, Dr. Adamczyk emphasized that this collaboration has already produced a number of publications (e.g., Enmark et al, Analytical & Bioanalytical Chemistry, 2019).
Dr. Konrad Bleicher, expert Scientist at F. Hoffmann-La Roche Ltd, started by explaining, that even though sulfur-based backbone modifications are the most relevant modifications in the space of RNA therapeutics, there is a need to go beyond the classical phosphorothioate to enhance stability and functionality of therapeutic ASOs.
In fact, Dr. Bleicher highlighted that, at La Roche, instead of going after the new phospho-mimics, the researchers have tried to dig out older chemistries to help design the oligonucleotides in use today.
Dr. Bleicher talked about the work of revisiting phosphorodithioates as phosphate bioisosteres, and its possible applications in pharmacologically active ASOs. Studies of Malat-1 RNA knockdown in different primary cell lines (fibroblast, skeletal muscle, bronchia epithelium) showed how the position of the PS2-linkage can alter the potency of phosphorodithioate modified gamer.
In fact, results from these studies are the main reason that at La Roche, researchers position dithioates in the flank regions, and not in other locations.
In relation to the in vivo potency of such modifications, La Roche studies have shown a significant in vivo potency increase for phosphorodithioate modified Malat-1 LNA gapmers in the heart tissue.
In relation to studies of ApoB mRNA target knockdown in vivo, comparing mixtures versus single compounds with or without PS2-modifications, the results have shown that stereodefined phosphorodithioate ASOs show the strongest in vivo performance at all time points measured. Also, PS2 modified ASOs show higher organ exposure and a superior metabolite profile.
Dr. Bleicher concluded his talk by saying that single PS2 at the 3’-end may substantially impact metabolite profile, with no indications for toxicity observed so far (in vitro & in vivo). Also, PS2-modifications are well tolerated in flank regions (LNA, MOE; 2’OMe), with in vivo efficacy demonstrated for various tissues (e.g., heart, liver).
Symmetry of non-bridging dithioates reduces chiral complexity; and stereodefined anti-ApoB phosphorodithioate shows superior in vivo efficacy, target organ exposure and drug metabolism profile.
Dr. Olivier Ludemann-Hombourger, Global Director of Innovation and Strategy at Polypeptide Group, started by explaining the in-house development strategy for the peptide manufacturing route.
Ideally the synthesis route is locked at early stage as to not affect the process performance at the commercial stage.
For that, the development needs to be an iterative process, with crude purity and critical impurities at the center of the upstream and downstream process flow.
There is a need to have predictive tools based on gathered experience, advance monitoring to collect data and modelling tools for a better understanding of the mechanisms and extrapolation.
Dr. Ludemann-Hombourger emphasized that sometimes, critical impurities cannot be avoided, leading to a challenging purification problem.
He gave the example of an industrial case study, where the analysis made with a TFA buffer showed that critical impurities co-eluded with the target peptide.
In order to overcome such problem in DSP optimization, Dr. Ludemann-Hombourger stressed there is a need to resolute all critical impurities with isocratic buffers for example, which can induce a repulsion mechanism at heavy mass overload, with improved thermodynamics where all impurities are isomers.
As such, an adaption and optimization of the chromatographic system and conditions, can help to identify the nature of critical impurities and resolve purification problems.
As productivity/yield are the key optimization criteria for process development, Dr. Ludemann-Hombourger emphasized the need to find the best compromise between both.
It is common practice to recycle side fractions; but there is also the possibility of improving resolution, by decreasing the loading, or the gradient slope, in order to increase the yield.
Of course, Dr. Ludemann-Hombourger stressed that performance cannot be undermined by a poor process robustness.
In relation to innovation opportunities, Dr. Ludemann-Hombourger highlighted the latest gradient multi-column concepts developed at ETH Zürich (MCSGP process) and at NOVASEP (GSSR process).
Even though these are more complex systems, these emerging technologies can lead to improved yields/purity, with higher productivity and lower eluent consumption and fraction recycling.
Dr. Patrick Brennecke, Technical Director of Bioanalytical Translational Science at Celerion, began the presentation by explaining the challenges for the development of a surrogate biomarker assay that would match the Quality Controls (QC) of the peptide hormone insulin.
Dr. Brennecke warned of kit-lot changes that could have an impact on the assays and undermine the results. He explained that a solution to this problem was the usage of a surrogate matrix buffer, which was critical and able to significantly reduce the lot-to-lot variation seen in insulin-depleted sera.
Also, the dilution of the sample buffer was able to abrogate the cross-reactivity seen with insulin analogues, making the measurements more reliable.
Next, Dr. Brennecke talked about Ghrelin, which, in a play with words, he said was “hungry for lower sensitivity”. This is because around 90% of study samples in their readings were Below the Limit of Quantification (BLQ) when using the usual kit-based total Ghrelin ELISA assay (validated in the range of 336-1600 pg/mL).
To circumvent this problem, Celerion developed a methodology using LC-MS/MS with an LLOQ of 50.0 pg/mL for detection of both Ghrelin species, acylated Ghrelin and des-acylated Ghrelin.
Using this methodology, the level of BLQ values went down from 90% to 27%; and allowed the detection of both forms of Ghrelin (acyl & des-acyl).
As such, having an LC-MS method in place is a plus, in comparison with the usual ELISA assays, which only quantify total Ghrelin, without discriminating both forms of this “hunger peptide”.
The final part of Dr. Brennecke presentation was about the future trends in ultra-sensitive detection of proteins and peptides.
He explained that many cytokines/chemokines, particular in chronic diseases (e.g., Psoriasis, Rheumatoid Arthritis), show small changes in a very low analytical range.
The new SIngle MOlecule Array (SIMOA) is able to detect proteins/peptides in the fg/mL-pg/mL range. For example, IL-6 levels analyzed at Celerion were only different between healthy and disease individuals when using the SIMOA method of detection.
Also, in the case of TNF-alpha, it could only be detected in several patient samples using this method. As such, at Celerion, SIMOA is now used routinely for the detection of low abundant cytokines and peptide drugs.
Dr. Andreas Kuhn, Senior Vice President, RNA Biochemistry & Manufacturing at BioNTech RNA Pharmaceuticals GmbH, begun by explaining that the selection of the best antigen against SARS-Cov-2 was made based on previous knowledge of other Coronaviruses, and targeted the spike protein.
Different variants were designed (both on amino acid and nucleotide - i.e., codon usage – level) and tested in multiple preclinical systems (in vitro expression data, antibody titers in animal models, pseudo-virus neutralization (pVN) assay in animal models).
The BioNTech strategy was to evaluate all three available RNA types: Uridine mRNA (uRNA), Nucleoside-modified mRNA (modRNA), and Self-amplifying mRNA (saRNA). The rationale for testing three mRNA types in all Phase I/II studies of the COVID-19 vaccine was the following:
With this in mind, BioNTech scientists designed multiple antigen variants using the SAR-CoV-2 S protein; and conducted preclinical evaluation, in order to bring the most active antigen into the clinic.
Preclinical/toxicology data was generated for approval of the clinical study; and “lightspeed” manufacturing of small-scale batches was started for fast entry into the clinic, in order to demonstrate safety, tolerability (i.e., low reactogenicity), and immunogenicity.
Next, clinical trials were simultaneously initiated with four vaccine candidates to accelerate development and identify the safest and most effective candidate for further development.
In parallel, there was preparation of larger batches for manufacturing of the candidate with the best output in the clinical studies; and, the identification of collaboration partners to develop and provide the vaccine worldwide (Pfizer, FosunPharma).
Dr. Kuhn emphasized that what made this process “lightspeed”, was the fact that BioNTech already had GMP-compliant RNA and LNP manufacturing processes with clinical experience for this specific technology.
This fact, together with early and constant interaction with the regulatory agencies, allowed for the first GMP batch to be available within 84 days after the first BioNTech internal project meeting. Not only that but, if a new variant was required (e.g., due to a mutated virus sequence), a new GMP batch could be available within half of this time.
Currently, a global Phase II/III with vaccine candidate BNT162b2 has been initiated with up to 44,000 subjects to be enrolled. As of Oct.14th, 37,000 have already been recruited.
If the data holds true, BioNTech and partners are hoping to fill for regulatory approval under an Emergency Authorization and provide a global commercial supply of COVID-19 vaccine.
NOTE: On November 21st, a week after Dr. Kuhn’s talk at TIDES Europe 2020, BioNTech and Pfizer have applied to the FDA for Emergency Approval of the new COVID-19 vaccine.
Dr. Jimmy Weterings, Principal Scientist of Oligonucleotide Chemistry at AstraZeneca, opened by defining a conjugate as a composite formed by the joining of two or more chemical compounds, which can improve the therapeutic window of the therapeutic oligonucleotide, by refining pharmacokinetics, biodistribution or a productive uptake of the nucleotide.
The target-delivery of oligonucleotides requires specific or enriched receptor expression on the target cell.
The ligands binding to the receptor needs to do so with sufficient affinity and selectivity, in order to promote the cellular uptake mechanism, ideally without activating signaling. Also, there is a need to escape the endosome in order to have transcript knock-down and functional response.
The selective ligand can be an aptamer, cholesterol, folic acid, an antibody, GalNac or even a Peptide (iRGD, cRGD, CPP), that is able to specifically bind to the receptor on the target cell.
Through solid and solution phase synthesis, it is possible to position the ligand using several conjugation options: (1) both 5’ and 3’ positions and 3’ of antisense strand in siRNA; (2) both 5’ and 3’ positions in ASOs; or, (3) internal position within siRNA (ss and as) or ASOs.
Whether using solid or solution phase synthesis, Dr. Weterings reminded us that it is important to use conditions that are compatible with both oligonucleotide and ligand.
Next, Dr. Weterings talked about the biological impact of ligand conjugation strategy, which can lead to enhanced targeting to cell/tissue of interest, improved uptake of the oligonucleotide, reduced susceptibility to nuclease activity, no need for transfecting agents and improved pharmacological activity/efficacy.
Different types of linkers can be used, like irreversible linkers (stabile linker type that does not release the targeting ligand from the conjugate); reversible linkers (labile linker type that under specific conditions can release the targeting ligand); enzymatically cleavable linkers (labile linker type that upon contact with specific enzymes can release the target ligand); and non-covalent linker (labile “linker” holding the conjugate in place via interaction, releasing the ligand upon specific conditions).
Finally, Dr. Weterings talked about oligonucleotide conjugation opportunities, like toxin-siRNA conjugates for Central Nervous System (CNS) delivery, giving the example of the Cholera toxin siRNA (CTB-CTA2-CTA1-siRNA construct).
Dr. Philip Gotwals, Global Head of Business Development and Licensing (BD&L) at Novartis Institute of BioMedical Research (NIBR) Inc., described how is team works with prospective partners to establish collaborations that are mutually beneficial and productive.
They remain actively engaged with collaborators to ensure a dynamic collaboration environment and maximise the probability of success.
The NIBR Business Development & Licensing team is flexible in structuring deals and aims to tailor collaborations to each partner’s individual needs.
Deal types include in-licensing, out-licensing/risk-sharing, research collaborations, options, equity investments and acquisitions in most therapeutic areas and technology classes.
From idea-generation and early discovery through clinical proof-of-concept (Phase IIa clinical trials), Dr. Gotwals emphasised that only by opening up to the world, can we have a better chance of fulfilling the promise to get the best medicines to patients efficiently.
