TIDES ebook - Analytical Strategies for Oligonucleotide and Peptide Therapeutics GATE
This TIDES eBook focuses on the challenges, strategies and technologies for measuring quality of oligonucleotides and peptides in therapeutic development.
Analytical Strategies for Oligonucleotide and Peptide Therapeutics
Introduction and Contents
Peptides and oligonucleotides have historically incurred control challenges in drug development due to their uniqueness and complexities.
With regulatory guidance still slow developing, the best way to avoid impurities is maintaining complete understanding of quality throughout qualifying raw materials, chemistry and downstream processing.
Sponsored by Asahi Kasei Bioprocess, this TIDES eBook focuses on the challenges, strategies and technologies for measuring quality of oligonucleotides and peptides in the processes of therapeutic development.
We’ll also explore advice from industry leaders and the uses of mass spectrometry and OPC platforms.
Jump to any article using the contents on the following page, or at any time using the Contents menu in the top left. There you can also download this eBook as a PDF.
1. The Industry's Biggest Challenges
We surveyed today’s professionals in the peptide, oligonucleotide therapeutics sector on their biggest challenges in meeting regulations in drug development.
The Industry's Biggest Challenges
An industry report
The Industry’s Biggest Challenges
In February 2021, we conducted a global survey of professionals working in oligonucleotide, peptide, mRNA and genome editing to find out how organizations in the industry are now working and what key strategies may have changed as a result of the COVID-19 pandemic.
Quality control and control of impurities has remained the biggest challenge for the respondents developing peptide therapeutics since our last report in 2019.
Delivery systems were the top mentioned by those working on oligonucleotides, a shift away from safety which was the top mention in 2019.
What are the biggest challenges in meeting regulations related to…
Top Response: Quality control
- "Efficacy, cost, patient adherence"
- "Oral delivery"
- "Long peptides = synthetic proteins, manufacturing at acceptable cost"
Top Response: Delivery systems
- "GMP procedures"
- "Scale and foreign dependency on raw materials"
- "Intracellular reach"
- "Evaluation of ADMET for longer nucleotides: Pharmacokinetic bioanalysis and immunogenicity for example"
- "Setting limits of impurity specifications"
Introduction to Analytical Control Strategies for Therapeutic Oligonucleotides
TIDES Training Series | September 14-16, 2021
Driving Efficiency and Ingenuity with an OPC Platform
by Chris Rombach, VP Sales & Marketing,
Asahi Kasei Bioprocess America
Driving Efficiency and Ingenuity with an OPC Platform
The regulatory approval of several oligonucleotides over the past five years – combined with the recent headlines around rapid COVID-19 vaccine development – has created significant interest in the oligonucleotide (DNA/RNA) segment of the scientific industry.
This includes everything from antisense oligonucleotides (ASOs) to lengthy gene transcript mRNA, as well as shRNA (small hairpin RNA), siRNA (small interfering RNA), CRISPR/Cas9 and anti-miR (anti-micro RNA).
The ability to rapidly create, study and formulate these nucleic-acid-based sequences and obtain authorization to provide timely therapeutic benefits to patients is more critical than ever.
Robust data analytics from each unit operation – as well as throughout the processing flow continuum – can allow for fewer batch comparisons, thereby decreasing the molecular development timeline, in addition to decreasing the molecule’s cost to market.
To achieve these advances, however, there may need to be changes to currently accepted equipment design and utilization mechanisms for nucleic acid production.
Many times, a preferred platform may be utilized for X operation, and yet another might be used for Y operation.
This preference can be driven by hardware, software, industry expertise, customer support, lead time/availability and/or design; but, regardless, can create unnecessary imbalances in data output.
The ideal arrangement would be to have a unified platform that could communicate with all equipment variations, allowing customers to choose any vendor without sacrificing core or regulatory type expectations for analytics capture and reporting.
Such a solution would entail a future-proof, open platform communication (OPC) setup that would allow scalability and the ability to cover many instrument types.
Labor Reduction, Accuracy, Remote Operation
In the therapeutic oligonucleotide synthesis unit operation, many current manufacturing practices rely on manual, personnel-dependent measurement of the packed column bed height which is then entered into the synthesis recipe by hand.
In an OPC environment, a bed height sensor on the column would automatically detect measurements and, due to its platform independency, then interface to convert this generic OPC request into a device-specific request for the synthesizer.
This could allow the synthesizer to automatically adjust the processing recipe and/or the appropriate chemical deliveries for charges that are correlated to column volume and/or bed height.
The result would be that the product impurity profile – and thus product safety and efficacy – has been maintained alongside the removal of potential human error.
An additional OPC benefit would be the integration and combination of controls that are used to create the chemical reagents necessary for successful therapeutic formation.
Do you believe an open platform communication (OPC) environment will be a critical component of next-gen oligonucleotide manufacturing?
Accurate composition and delivery, as well as the use of analytics to confirm compositional expectations of these reagents, will ensure product consistency and process accuracy.
New technological advances are quickly being made in nucleic acid chemistry, whether it be in production and optimization, new chemistries, delivery and uptake for the patient, or clinical efficacy.
These rapid developments may not be easily incorporated into a standalone equipment platform due to potential secondary and tertiary consequences, thus requiring lengthy testing and qualification assessment.
An OPC setup also allows for the ability to monitor and easily operate hardware remotely, or via a mobile device, enabling resource efficiency.
This could be financially beneficial for smaller sites that cannot afford to invest in additional personnel shifts, yet possess the demands to run operational activities during off-shift hours.
The use of an OPC-based platform would remove the need for more traditional techniques, such as remote desktop access. The incorporation of OPC could also allow for simultaneous user access, which may not be feasible in a remote desktop environment.
Driving Quality and Productivity
Distinct and detailed analytics throughout the oligonucleotide process can provide greater assurance of product quality, product safety, process accuracy and quick identification of areas requiring process improvement.
The monitoring of multiple data streams during each coupling cycle can help identify variable output data, even when all appropriate processing parameters have been set/controlled.
Product quality that can be understood early in the process, combined with process understanding founded in analyzed data, can help with planning downstream operations in a manner that ensures the final product is of proper safety, identity, strength, purity and quality (SISPQ– 21 CFR 210.1(a)).
For example, if a 21-mer (base length) oligonucleotide has 98.5% efficiency for each coupling, the resulting theoretical purity ceiling will be ~72.8%; but if that efficiency is improved to 99% for every base coupling, the theoretical purity ceiling will now become ~81.0% (see Figure 1).
Although this 0.5% change may not appear significant from an empirical value perspective, proper analytics can help assure that this migration in efficiency can make a significant positive alteration to the attained purity.
Not insignificantly, this purity improvement could result in fewer executed unit operations or a lower cost of goods.
In such a case, a conjugated oligonucleotide may not require the use of a chromatography/purification operation if the purity is high enough, therefore earlier recognition of the product purity might drive certain processing decisions.
OPC-driven analytics may also provide the possibility of using data to determine execution of maintenance for more efficient downtime management.
Most often, time-based execution of maintenance (every 3 months, 6 months, etc.) frequencies have been outlined or prescribed in the production environment to help ensure that production equipment is running as smooth as possible for as long as possible.
The use of analytics connected and based upon a central platform could provide the data to demonstrate a more accurate justification/timing to perform any necessary maintenance ("predictive” maintenance).
The use of analytics for process accuracy can be beneficial to confirm product safety (against a clinically accepted impurity profile) and can also assist with product/process investigations should there be a need to compare against historical batches.
Sensors that can monitor delivery flow rate and delivery volume with a high data frequency attainment rate can provide not only significant data, but also assurance that the process was executed as expected.
OPC Avoids ‘Standalone’ Pitfalls
A standalone system that is not OPC compliant/compatible could inhibit industrywide advancement as the incorporation of new sensors or new technology would require assurance that the existing hardware or software is not impacted by the upgrade.
Further, standalone systems likely contain proprietary methods for accessing “behind the scenes” analytics used to help diagnose, or preventively identify, an equipment-related issue. OPC-ready systems should allow quicker analysis and diagnosis of any imminent issues.
The synthetic creation of oligonucleotides requires the integration of many systems, including those that demonstrate quality.
Many of the quality systems are integrated into the regulatory commitments to ensure that product efficacy and safety are maintained. While there is heavy focus on product efficacy, and rightfully so, process efficacy would most certainly benefit from an OPC-type design.
Analysis of Antisense Oligonucleotides Using Ultra-high-resolution Mass Spectrometry
A presentation by Jingshu Xu, PhD, Senior Scientist in Mass Spectrometry, AstraZeneca
Analysis of Antisense Oligonucleotides Using Ultra-high-resolution Mass Spectrometry
This presentation was given by Jingshu Xu, PhD, Senior Scientist in Mass Spectrometry, AstraZeneca at the virtual TIDES Europe 2020 and summarized by David Orchard-Webb.
Dr. Jingshu Xu talked through AstraZeneca’s approach to MS data processing for oligos.
To support the new modalities portfolio and handle more complex molecules, AstraZeneca has a range of MS capabilities. For oligo analysis, a number of mass spectrometers offer various resolutions.
They offer different modes of ionization, dissociation, and different types of mass analyzers. The Waters Q-TOF comes with a mobility, for example, giving 3D resolution. The Thermo Fusion Orbitrap is a trap read that comes with a choice of three dissociation methods; CID/HCD/ETD.
The new modalities are also supported by the Bruker UltrafleXtreme, a MALDI-TOF/TOF instrument, and ScimaX which is an ESI MRMS instrument that comes with extremely high resolution. Both ESI and MALDI source for versatility offering further flexibility.
Most modern pharmaceutical oligonucleotides are resistant to enzymatic digestion precluding the typical DNA sequencing methods. The principle of sequence confirmation by MALDI-TOF or LC-HRMS/MS is quite straightforward.
The intact molecule is ionized and fragmented using available dissociation methods and the fragments ions are measured by the mass analyzer. The sequence can be deduced by working out the differences between the fragment ions.
For an efficient sequence confirmation workflow, AstraZeneca wanted a simplified MS method that provides good sequence coverage through comprehensive fragmentation but also a more streamlined way to deal with the complex output data.
As opposed to the LC-HRMS method, fragmentation in source was coupled with single stage MS to take advantage of the ultra-high resolution of the Orbitrap. To deal with the complex data analysis, an in-house automated data process and interpretation workflow was developed.
Automated steps include the calculation of expected fragment ions based on the McLuckey fragmentation scheme. It also models the isotopic patterns to allow the choice of either monoisotopic or the most abundant mass.
The hit list from the MS is searched against the calculated theoretical fragment ions in an exhaustive manner for matches within a given parameter.
The specialist is required to focus on the methods with no match in the automated search. A sequence coverage plot has been created to visualize the output in a simple yet informative manner.
Automated data processing has allowed fast data interpretation through exhaustive search and systematically applied filtering, which in turn gives consistent spectrum annotation and also consistently informative reports for a large amount of data in a very short time.
Furthermore, the visualization reveals sequence coverage, and fragment ion distribution from each experiment at a glance.
To demonstrate sensitivity, switch sequences of Danvartirsen were made by swapping two bases around in position. All switched sequences were analyzed using the simplified LC-MS method.
From the MS, differences were identified, especially in the center region of the spectrum.
The hit list from each mass spectrum was searched against the calculated theoretical fragment ions of the correct Danvartirsen sequence.
Essentially, this is an alignment of how much the spectrum matches that of the correct sequence. In this way, it becomes obvious which fragment ions do not match those that are expected of the correct sequence, which gives a good indication of the position of the base mismatch.
This approach was used to quantify the level of deamination impurity in Danvartirsen. In Danvartirsen, there are three sites with a high probability of deamination. Deamination is effectively a loss of ammonia in exchange for water resulting in a mass shift of one dalton.
By fragmenting the molecule, small fragment ions (around 700-1500 dalton) that contain those deamination sites individually can be monitored and mass-charge shifts detected.
Xu would always start with more than one fragment ion to monitor for each deamination site and also use site specific deamination impurity standards to aid the quantification itself.
In a recent study evaluating the risk associated with terminal sterilization, the described method was used to quantify lower levels of deamination.
From the result, an intriguing observation was made: the terminal methylC residues showed lower levels of deamination than the one in the middle of the sequence.
Increasing the throughput of data analysis is a critical part of achieving an efficient analytical platform for oligonucleotides and AstraZeneca is currently in the process of further developing automated data processing workflows for oligos. Quantification of degradation impurity is another area of focus.
EMA guidelines require a risk assessment for terminal sterilization, even though deamination is a low risk for aseptic processes. AstraZeneca was able to quantify low levels of deamination impurities using the newly developed method.
Having a sensitive and site-specific quantification method also provides an exciting opportunity to investigate the site-specific reaction kinetics and formulation parameters in the future. Finally, in-source fragmentation-based methods are also attractive for chemical ID tests.
Analytical and Regulatory CMC for Oligonucleotide Therapeutics
A presentation by Thomas Rupp, Owner & Principal, Thomas Rupp Consulting UG
Analytical and Regulatory CMC for Oligonucleotide Therapeutics
David Orchard-Webb looks back on the highlights of a popular presentation given by Thomas Rupp, Owner & Principal, Thomas Rupp Consulting UG at TIDES Europe 2020.
Thomas Rupp talked about what makes the CMC of oligonucleotides (oligos) so unique, the regulatory background and the origin and the nature of impurities.
Vitravene oligo drug established a new class of therapeutics in 1993. From a regulatory standpoint they are chemical entities, but they are bigger than typical small molecules.
They are large polyanionic compounds that carry features of both typical drugs and biologics. They are produced by chemical syntheses which is a predictable, but impure, process.
Like biological entities, oligos are difficult to fully characterize because of their molecular composition and because of their heterogeneity.
Since 1993, the US FDA has regulated oligos as big small molecules and not as biologics. That means, in the US, most new oligo drug applications go to the Center for Drug Evaluation and Research (CDER), and in Germany, the equivalent is the Federal Institute for Drugs and Medical Device (BfArM).
There are exceptions which may be received by the Center for Biologics Evaluation and Research (CBER) in the US or the Paul-Ehrlich-Institut (PEI) in Germany.
For example, if oligos are used as an adjuvant in a vaccine or if they have an aptameric structure which is not sequence dependent. There are no international guidelines or specifications for control strategies of impurities.
Like synthetic peptides, oligos were excluded from the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines Q3A & Q3B, which refer to impurities in drug substances (DS) and drug products (DP), and the Q6A specifications for new DS and DP.
They are also excluded in the foreword of the ICH guidelines. As there are no formal guidelines for therapeutic oligos on limits and thresholds of impurities, they are discussed with the respective regulatory authorities on a case by case basis.
Such discussions are based upon the safety and class complexity of the molecule and the formulation. Sponsors have the flexibility of adapting guidelines for other entities (synthetic peptides for example, biologics, antibodies) with appropriate justification for the oligo.
In terms of how the usual ICH Q3A requirements on impurities can be extrapolated to oligos, Rupp focused on starting materials, byproducts, and degradation products.
Dealing with oligo impurities requires a thorough understanding of the quality of the raw materials, and the chemistry during synthesis and downstream processing.
Understanding the stability of the oligo compound during the syntheses, downstream processing and during the storage is important. Degradation pathways should be known.
A 2017 white paper by the oligo safety working group (OSWG) suggested to categorize impurities into four different impurity classes and assess safety and potency only for impurities classified as critical and non-natural. According to Rupp, this whitepaper is well recognized by drug sponsors, and agencies when defining CMC control strategies.
The publication contains a useful decision tree for impurity classification. Accordingly, class I impurities are impurities that are also major metabolites. The structure and the sequence are the same as the parent compound.
Class II impurities contain only structural elements found in naturally occurring nucleic acids. Class III; impurities that are sequence variants of the parent oligo.
That is base deletions or additions and also deaminations. Class IV are impurities that contain structural elements not found in the parent oligo or in naturally occurring nucleic acids.
These are base modifications, backbone modifications, depurinated species, and unidentified impurities and this is the only class that requires a safety assessment if the impurities are above the threshold.
An impurity identification threshold of 1% and a qualification threshold of 1.5% is suggested. Typically, industry uses a 0.5% threshold for identification and 1.5% for the qualification.
Rupp recommends, firstly, to avoid impurity formation, if at all possible, because not all impurities are easily purified out. In a good control strategy, you would use orthogonal analytical methods to support identification and separation of different impurities.
If critical impurities are detected above the threshold level, the performance of supporting in vitro studies to allow proper classification and comparison to the parent compound and to assess the safety in vitro and in vivo are suggested.
It is a good idea to trace back identified impurities to the relevant process step and perform selective post-development to avoid the formation of these impurities.
ICH Q3 states that any molecule that is different to the parent molecule (full-length oligo) is categorized as impurity. Oligos are excluded from ICH Q3A (impurities in DS) and IHC 3B (impurities in DP) and there are no explicit guidelines on process-related impurities available. According to the OSWG classification system, class IV impurities require a safety assessment.
Analysis by high-resolution LC-MS can allow tracing the origin of each impurity back to the respective process step and allows for successive process optimization.
Capaldi, Daniel, et al. “Impurities in Oligonucleotide Drug Substances and Drug Products.” Nucleic Acid Therapeutics, vol. 27, no. 6, 1 Dec. 2017, pp. 309–322., doi:10.1089/nat.2017.0691.