Susan A Darling, Senior Director, CE and Biopharma, SCIEX and Todd Stawicki, Senior Global Marketing Manager, Biopharma, SCIEX examine advanced analytical technologies can help ensure that biopharma candidates are commercialisable.
Developability in the biopharmaceutical industry relates to the difficulty of making early candidate molecules with attractive biological activities into commercialised and marketable therapies. The ‘developability gap’ is the degree of uncertainty when the information needed to make such assessments is lacking at earlier stages of a process. While the developability gap is not new, the level of uncertainty and risk associated with biopharmaceutical development has increased measurably in recent years as the pressure to bring only first-in-class or best-in-class drugs to market has risen. Furthermore, many of these therapeutics are increasingly complex compared with their predecessors with fusion proteins, multi-specific antibodies and antibody-drug conjugates constituting a larger proportion of therapeutic portfolios. To close this gap, developers of biotherapeutics need more information about their molecules far earlier. This information will allow them to make confident and informed decisions about how to prioritise or optimise their best candidates. Doing this earlier will help minimise costly time delays, or late-stage program failures.
Why biopharma struggles with the developability gap
The developability gap for small-molecule candidates is generally minimal because there are numerous analytical technologies available for rapidly and cheaply gathering information to generate highly complete descriptions. The same is not true for proteins, antibodies, viral vectors, cells and other highly complex biologic drug substances. While there are many technologies for describing different properties of biomolecules, arguably none exist that rapidly provide sufficient depth and contextual breadth of analysis at a price point that is practical for widespread use during early development. There are many different technologies for describing different properties of protein biomolecules. There is arguably no single tool that provides sufficient depth and breadth to fully describe a protein at a price point that is practical for widespread use during early development. Therefore, scientists unfortunately need to use many analytical technologies at the same time. Integrating and resolving the diverse data generated by these various technology platforms presents an added challenge.
As a consequence, biotherapeutic programs must proceed at a certain degree of risk due to the uncertainty that exists with respect to the molecular properties of candidate molecules. Lack of access to this information can mask potential signals that could predict later issues with formulation, scale-up, manufacturing, transferability or even safety and efficacy.
A need for speed and quality
At the same time the complexity of biologic drug candidates has increased exponentially, development timelines are under pressure to continue shrinking. Not only are accelerated approval pathways for novel medicines being leveraged at a growing rate, the rapid approval of vaccines and therapeutics for Covid-19 has created a vision for developers to create drugs to progress to commercialisation at ‘pandemic speed’. With shorter timelines and greater complexity comes higher risk. Failure of a late-stage batch represents not only the cost of the material, but lost time under patent protection and the possibility of missing the window for introduction of a first-in-class product. Even for regular mAbs there are challenges.
With more variants to understand and less confidence in the techniques used to analyse them because they were originally developed for proteins, novel modalities present even more challenges. In fact, the expanding chemical space for biologic drugs makes the developability gap more relevant than before. The risk of late-stage failure increases if this information is not obtained early on. Understanding these molecules early in the process is important because much is not known about where they will be made—in a large central plant, a smaller, regional, modular facility or within a hospital.
To develop robust manufacturing processes, it is first necessary to fully characterise candidate molecules to understand the structural differences between variants and how those differences impact pharmacological activity. Then the process design space must be explored to understand how changes in production conditions can impact the distribution of variants obtained. Unfortunately, many assays for biotherapeutics are indeed functional assays that evaluate a biophysical behaviour rather than molecular characteristics and structural features. Using such functional assays, it is possible to miss detectable structural features that do not impact the functional test but could be very good predictors of later-stage issues.
A fundamental aspect of biopharmaceutical drug development is the need for the data submitted in an Investigational New Drug (IND) to match the data containing in the Chemistry, Manufacturing and Control (CMC) section of a Biologics License Application (BLA). Any significant changes to the molecule when reformulating during dosing studies or upon scale up of the manufacturing process will create the need for bridging studies, leading to delays. A candidate can fail even if it has a good toxicity profile and excellent efficacy as demonstrated by clinical endpoints if it cannot be practically manufactured. It is essential to know upfront that a candidate has good developability and manufacturability properties. In addition, drugs receiving breakthrough therapy and other designations for which the approval process is dramatically accelerated have the potential to pivot to commercial production after Phase II (with Phase III trials performed after launch) if the study results are favourable. It is essential to know that such a biologic drug candidate is commercialisable at square one and that the process used to produce the clinical materials is also commercialisable. Companies are thus faced with the challenge of addressing the developability gap early on and from many perspectives. Formulation challenges can arise if the drug substance is not stable in the chosen buffer or aggregation occurs at the high concentrations ultimately selected for optimal dosing. Efficacy and safety issues can arise if the molecule degrades under physiologic conditions. To better understand the structural liabilities that could impact commercialisability, there has been a shift in the industry toward conducting stability studies much earlier in the development process using a range of conditions, including not only different buffers but the biological matrix, as well as different concentrations that could be used for the final product. Developability assessment strategies for both vaccines and liquid biologic formulations have been proposed. The former involved a two-step developability assessment workflow to screen variants of recombinant protein antigens under various formulation conditions to rapidly identify stable, aluminum-adjuvanted, multi-dose vaccine candidates1. The latter involves performance of a ‘manufacturability’ assessment by applying quality-by-design (QbD) principles to identify preliminary critical quality attributes (pCQA) and screen multiple candidate molecules with respect to the stability of these pCQAs upon storage and when exposed to relevantstresses2. Such statistical work early on in formulation development pays dividends, including the ability to develop the optimum formulation.
The use of less-sensitive assays in early-staged evelopment projects can also have a negative consequence when processes are scaled. Many impurities and/or variants are often present at very minute quantities at earlier scales but not detected with the less-sensitive assays typically used at that stage. Indeed, many biotherapeutic molecules that are very functional and well-behaved in the preclinical phase when produced in batches of a few litres can have issues when produced for clinical trials at larger scales (batches of a few hundred to several thousand litres). Glycosylation patterns, deamidation sites, isomerisation of amino acid residues etc can manifest in larger cell-cultures. The higher quantities of such variants obtained at larger scale may however, impact safety, lead to lower yields or create downstream purification challenges. In some cases, the variants or impurities may not be removable or the cost to remove then too high, rendering the product uncommercialisable. Alternatively, scale up of a process may result in an acceptable product, but one that is considered by regulatory authorities to be different from the original. Genzyme faced this issue in 2008 when the company scaled up a 160-L process for its approved Myozyme (alglucosidase alfa) treatment for Pompe disease to 2000 L3. The FDA has determined that alglucosidase alfa produced at the 160 L and 2000 L scales should be considered as two separate products because of comparability differences and Genzyme had to apply for a separate BLA for the product produced at the 2000-Lscale. This example highlight show important it is to fully characterise the variants of a biologic drug candidate before filing an IND. Although with limited resources, small and emerging biotechs are typically not in position to invest heavily in strategies for addressing the developability gap early on, many large biopharmaceutical companies have recognised the need to find solutions for this issue. One example is Novartis, which in 2014 published an article describing its Integrated Biologics Profiling concept, which was intended to de-risk the development process by combining cell line development, purification, biophysical characterisation, and in vivo fitness assessment activities into one group4. Investment in these resources in the early stages of development projects “enabled better candidate selection decisions at the R&D interface based on biology and developability data.”
Analytical instrumentation and techniques have continued to evolve. New solutions have been introduced, or are under development, to help manufacturers rapidly obtain data with more confidence.
Imaged capillary isoelectricfocusing – mass spectrometry
One example is icIEF-MS (imaged capillary isoelectric focusing – mass spectrometry) for analysis of biotherapeutic charge variants. Evaluation of charge variants is a regulatory requirement and essential to determining the developability of candidate molecules.
Currently, samples are subjected to cIEF for peak separation followed by ion-exchange (IEX) chromatography to identify the different variants. Because IEX does not have the same resolution as cIEF, the fractions collected following IEX must be reinjected on a capillary electrophoresis (CE) system to understand how they correlate. The relevant fractions are then subjected to peptide mapping. Preparing samples and performing the separations and analyses takes a great deal of time—this can be months. And because IEX methods are product-specific, a different method must be developed for each candidate, with the possibility of having 10s of candidates to analyse.
All collected data must be synthesised so that modifications on each candidate molecule can be evaluated with respect to their ability to impact function. With icIEF-MS, the desired information about charge variants can be obtained in a single day. The tremendous amount of time saved can lead to a reduced development timeline while also enabling more thorough analysis of the impacts of process changes on structural characteristics and so on.
Multicapillary CE-SDS-MW analysis
CE-SDS analysis for molecular weight confirmation and purity analysis is currently considered one of the ‘gold standards’ for protein therapeutic characterisation all the way from discovery through product release. While it is a very important method, it can be time consuming – particularly when there is method development required for new types of protein therapeutics.
Electron-activated dissociation (EAD) fragmentation
Many critical quality attributes such as disulfide bonds and amino acid isomerisations can have profound impacts on the quality and safety of biotherapeutics. These molecular features have traditionally been extremely difficult and time consuming to analyse with traditional mass spectrometry techniques such as collision-induced dissociation (CID) or electron-transfer dissociation (ETD). A new technique that offers insights that could not be obtained previously is electron-activated dissociation (EAD) fragmentation coupled with Zeno trap technology. This technology offers unambiguous detection and identification of both expected and scrambled disulfide bonds and many types of deamidations and isomerisations. Using this technology enables earlier and better process development optimisation of biotherapeutics due to deeper and more thorough characterisation of product quality attributes. EAD provides reliable results with the confidence needed to answer questions about developability and manufacturability, even for PTMs that have traditionally been difficult to identify with assurance. EAD affords drug developers another way to confidently interrogate the structure of biopharmaceutical candidates at a level of detail not previously possible. This information can be used to weed out undesirable candidates that might otherwise have been thought to be acceptable, thereby de-risking development and preventing late-stage failures.
De-risking development for success
Having better information sooner is the key to de-risking development; the ability to make confident decisions in the early phase enables a right-first-time approach to biopharmaceutical drug development. Indeed, the overarching goal when addressing the developability gap is to de-risk the drug development process and make it easier for drug developers to link structure to function. Tools that enable better evaluation of the development risk—and ideally resolve that risk—earlier in the process are incredibly valuable. Analytical techniques such as icIEF-MS, multicapillary CE-SDS-MW analysis and EAD fragmentation coupled with Zeno trap technology can help move development forward.
Along with other strategies such as early-stage stability testing in matrix and at high concentrations, these techniques help de-risk biopharmaceutical drug development. The ability to rapidly gain insights into molecular characteristics, structural features and potential scalability challenges and formulation issues at the early development stage dramatically increases confidence that candidates selected to move into the clinic will be commercialisable and reduces the chance of late-stage failures. Continuing advances in analytical technologies will help further address the developability gap and de-risk drug development. Ideally some of these methods will enable evaluation of multiple critical quality attributes in a single assay. New solutions for rapidly aggregating and analysing the increasing quantities of data generated during early-phase development will also help drug developers identify trends and correlations and better visualise results.
Volume 23 – Issue 3, Summer 2022
About the authors
Susan Darling is the Senior Director of capillary electrophoresis and capillary electrophoresis – mass spectrometry product lines at SCIEX. She is responsible for strategic direction of the CE and CE-MS at the company as well as new product and workflow development.
Todd Stawicki is a Global Marketing Manager for MS Biopharma at SCIEX. He is passionate about empowering customers with the most innovative and effective LC-MS and CE-MS methods available to achieve their scientific goals. He has over 15 years of experience as a research scientist at several pharmaceutical and biopharmaceutical companies and as a LC-MS applications scientist at SCIEX.
1: Nishant Sawant et al., “Rapid Developability Assessments to Formulate Recombinant Protein Antigens as Stable, Low-Cost,Multi-Dose Vaccine Candidates: Case-Study With Non-ReplicatingRotavirus (NRRV) Vaccine Antigens,”Journal of PharmaceuticalSciences, Volume 110, Issue3, March 2021, Pages 1042-1053. https://doi.org/10.1016/j.xphs.2020.11.039 .https://www.sciencedirect.com/science/article/pii/S0022354920307759
2: Bernardo Perez-Ramirez et al, “Approaches for Early Developability Assessment of Proteins to Guide Quality by Design of Liquid Formulations” in Quality by Design for Biopharmaceutical Drug Product Development, April 2015, Springer, Ed. by Feroz Jameel, Susan Hershenson, Mansoor A. Khan, Sheryl Martin Moe. https://www.researchgate.net/publication/279931481_Approaches_for_Early_Developability_Assessment_of_Proteins_to_Guide_Quality_by Design_of_Liquid_Formulations
3: Genzyme, “FDA Advisory Panel toDiscuss Genzyme’s Myozyme BLAon Tuesday: Meeting Will Focus on Clinical Data Supporting 2000L Process,” Press Release, Oct 17,2008. https://www.fiercebiotech.com/biotech/fda-advisory-panel-to-discuss-genzyme-s-myozyme-bla-on-tuesday
4: Thorsten Lorenz, PhD et al.,“Developability Assessment of Biologics by Integrated Biologics Profiling,” AmericanPharmaceutical Review,August 29, 2014. https://www.americanpharmaceuticalreview.com/Featured-Articles/167439-Developability-Assessment-of-Biologics-by-Integrated-Biologics-Profiling/