Bortone, Osvaldo (2023) Design of a microfluidic platform to study the stability of biologics. [Tesi di dottorato]

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Abstract

BACKGROUND: Biologics, such as protein-based injectables, can improve clinical outcomes in treating challenging diseases. Despite their potential, the use of biologics in clinics is still facing some obstacles due to various aspects. Among these, the major concern is represented by their immunogenicity, i.e. the propensity of the biotherapeutics to induce the body to mount an immune response aimed at modifying or neutralizing the therapy efficacy, or triggering severe side effects. The immunogenic behavior of protein therapeutics lies in their natural propensity to destabilize in response to external physico-chemical stimuli and form protein aggregates that are recognized as dangerous from the body. With the aim to preserve their therapeutic effect, the stabilization of proteins in solution against external stressors is typically attempted through the addition of co-solutes, named excipients. The final solution including the latter and the protein active ingredient (API) is named formulation. The latter, also named drug product (DP), can be defined as a multicomponent solution having challenging chemical (high API concentration and complex protein-protein/excipient-protein network of interactions) and rheological (high viscosity) properties. In order to be safely administered to patients, the proper formulation composition intended to stabilize the API as best as possible is determined through a formulation development study, i.e., the process where major factors threatening the API stability are identified and their impact mitigated through the modulation of excipients nature and combination. In general, in a formulation development process a set of candidate DPs, having same API but different combinations of excipients, are exposed to a number of physico-chemical stimulations (e.g., high temperature or agitation). These stresses are intended to accelerate the API degradation, so as to rapidly generate data about therapeutic protein stability behavior. The different compositions of candidate formulations cause them to exhibit dissimilar resistance to the aforementioned stimulations. Consequently, it is possible to identify a formulation ranking where DPs performing the worst are discarded, while those having the best stability behaviors are eligible to further investigations, manufacturing, (pre)clinical trials and potential marketization. AIM OF THE THESIS: While there is a pressing requirement for more robust formulations to precisely address patients' needs and enhance clinical outcomes, the stabilization of therapeutic proteins proves to be a challenging task due to the elevated number of instability sources and the complex physico-chemical nature of formulations. Current in-batch approach result to be low-throughput and expensive. Plus, they fall short of adequately simulating destabilizing conditions and do not provide the chance to combine stress sources - a common occurrence in real formulation lifecycles. Consequently, the protein stability behavior is only partially caught and a significant gap still exists in the scientific understanding of the intricate connections among therapeutic protein stability, physico-chemical destabilizing sources and potential mitigation strategies. To clarify this correlation, unconventional technologies are needed to enable cost-effective and systematic formulation screening against unexplored instability sources, such as high intensity hydrodynamic forces typical of manufacturing process conditions and combination of stresses. The integration of this information to the knowledge from current formulation development approaches could lead to a comprehensive understanding of the therapeutic biomolecule behavior, aiming to set more appropriate formulation studies as well as suitable molecule-related manufacturing process parameters. In light of the needs reported above, the present work aims to study the physical stability of therapeutic proteins in high concentration and viscous pharmaceutical formulations through the adoption of a miniaturized strategy based on the microfluidics. In fact, while the microfluidics has been successfully applied to the characterization of diverse aspects of therapeutic protein, its employment remains limited to diluted biomolecule solutions, with no clear focus on industrial context, where the main emphasis is on highly concentrated and viscous multicomponent formulations. In particular, we report the design and the development of an automated and programmable modular microfluidic platform able to apply unconventional physical stimulations to pharmaceutical formulations spanning a wide range of component concentration and viscosity. Specifically, it is intended to expose therapeutic formulations to unconventional thermal stress, mechanical stimulation (manufacturing-relevant shear rate levels) or a combination of them in an extremely controlled and reproducible manner. The platform is properly thought to automatically guide candidate formulations through predefined isolated or combined stress pathways, in order to induce API instability. METHODS: The microfluidic platform was thought to present a modular logic, including a pumping (syringe pumps) and stress modules (thermal and mechanical (shear) stress). Each stress module consists of a (thermal or mechanical) stress station and a recirculation line, properly intended to control the exposure time of formulation to the stimulation. Particularly, for the stress station, suitable microfluidic chip geometries were designed, characterized through numerical simulations and fabricated by machining polymethyl methacrylate (PMMA) through a micromilling process. For the recirculation lines and other fluidic path connections, automatic and programmable parts (Peltier cell, valves and pumps) were selected from the market on the basis of the operation requirements and the nature of processed fluids. Microfluidic chip geometries and process parameters were studied through simulative and experimental approaches to offer platform flexibility in terms of viscosity operational range, temperature control and shear rate thresholds. Highly concentrated (120 mg/ml) nanobody (nAb) - based formulations with different excipient combinations were thermally stimulated ( at 49 and 52 °C) close to the protein melting temperature through the microfluidic platform over different stimulation time (2 and 5 min). Its ability to determine aggregation was characterized via size exclusion high performance liquid chromatography (SE-HPLC). Results were compared to a well established miniaturized batchwise approach, through which the same formulations were thermally stressed at the same conditions. Additionally, the formulation ranking returned by microfluidics and small volume miniaturized stimulation was compared to those coming from long term standard pharmaceutical protocols, i.e., storage (5°C) and accelerated (25 °C) stability studies. Same formulations were subjected to isolated high shear levels (5500, 8000 and 20000 s-1) for different stimulation time (single passage through microfluidic shear station, 2, 5 and 10 min). Also, the formulations were subjected to conventional agitation protocols (300 RPM, 3 days). The effects of such mechanical stimulations were characterized by conventional analytics (SE-HPLC) and a quasi-real-time analysis (soon after the stimulation) based on dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FTIR). Additionally, the ability of the platform to combine stresses was demonstrated by subjecting a nAb-based formulation to thermal-mechanical and mechanical-thermal serial stimulation. Destabilization induced by the combination of solicitations were characterized through the same quasi-real-time analysis approach set up for the microfluidic mechanical stress investigation. RESULTS: The microfluidic platform exhibits high flexibility, as enables the possibility to process fluids spanning a wide viscosity range ([1;20] mPaxs) in a vast isothermal window ([25; 80]°C) and in a broad mechanical shear rate interval ([5500; 20000] s-1). The investigation conducted on the thermal stress led to demonstrate the microfluidic platform to express precise isothermal control with irrelevant temperature fluctuations. The comparison with well-established small-volume batch-wise thermal stress approaches showed the microfluidic platform to determine similar destabilization on nAb-based formulations. Additionally, the same formulation ranking obtained upon microfluidic thermal stress is comparable with the ones obtained through conventional storage and accelerated stability protocols. The experimentation of mechanical (shear) stress demonstrated the ability of the platform to induce dynamic/transient structural and colloidal instabilities. While conventional SE-HPLC was not able to detect any aggregation trend, the quasi-real-time analytical approach pointed out variations of biomolecule physical properties with different impact, depending on the stimulation intensity and formulation composition. Surprisingly, for some formulation compositions, more impactful destabilization might occur at lower shear rate thresholds, indicating how the excipient-protein interaction can be sensitive to specific hydrodynamic conditions. Additionally, the coupling of conventional agitation protocols to quasi-real-time analysis did not return any relevant trend. This confirmed the inadequacy of the agitation test to probe formulation robustness with respect to high hydrodynamic forces typical of manufacturing processes. Lastly, the versatility of the platform was shown by conducting serial thermal-mechanical and mechanical-thermal solicitations. Interestingly, the quasi-real-time analytical strategy highlighted unobvious formulation behaviors. In fact, it came out that the commutability of the stresses (thermal prior to mechanical and vice versa) was not verified. In other words, the order in which the stresses are applied might completely change the magnitude of structural and colloidal destabilizations. DISCUSSIONS: Although the microfluidics is often related to process scaling-down intended for cost reduction, in this application the microfluidic platform has proven to be much more than that. In fact, the predictability of transport phenomena in a microfluidic context enables a better correlation of stress sources and biomolecule instability behavior. Additionally, thanks to the possibility to subject DPs to unexplored stressors, the collection of novel and alternative instability information, that cannot be caught by conventional stress (e.g., agitation) and analytical protocols, can be achieved. Thus, the integration of such an approach to the current practice might broaden the knowledge of protein stability behavior at early development stages, gathering meaningful indications for later phases of a drug product lifecycle (e.g., manufacturing). This technology has the potential to shift the paradigm from a downstream corrective mode to an upstream testing approach and steer the development of biologics towards a more quality-oriented perspective, with the possibility to influence clinical outcomes.

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