Protein Aggregates in Inhaled Biologics: Challenges and Considerations.

pmc J Pharm Sci J Pharm Sci 3815 pheelsevier Journal of Pharmaceutical Sciences 0022-3549 1520-6017 pmc-is-collection-domain yes pmc-collection-title Elsevier - PMC COVID-19 Collection PMC9927828 PMC9927828.1 9927828 9927828 36796636 10.1016/j.xphs.2023.02.010 S0022-3549(23)00063-1 1 Special Topic Commentary Protein Aggregates in Inhaled Biologics: Challenges and Considerations Ibrahim Mariam a Wallace Ian b Ghazvini Saba a Manetz Scott c Cordoba-Rodriguez Ruth d Patel Sajal M. a ⁎ a Dosage Form Design & Development, Early-Stage Formulation Sciences, Biopharmaceuticals Development, R&D, AstraZeneca, Gaithersburg, USA b Clinical Pharmacology & Safety Sciences, Respiratory & Immunology, Neuroscience, Vaccines & Immune Therapies Safety, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden c Clinical Pharmacology & Safety Sciences, Respiratory & Immunology, Neuroscience, Vaccines & Immune Therapies Safety, BioPharmaceuticals R&D, AstraZeneca, Gaithersburg, USA d Regulatory Affairs, Chemistry, Manufacturing and Controls Regulatory Affairs, Oncology R&D, AstraZeneca, Gaithersburg, USA ⁎ Corresponding author. 5 2023 14 2 2023 112 5 428911 1341 1344 1 11 2022 9 2 2023 10 2 2023 14 02 2023 15 02 2023 19 04 2023 © 2023 American Pharmacists Association. Published by Elsevier Inc. All rights reserved. 2023 American Pharmacists Association Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Pulmonary delivery is the main route of administration for treatment of local lung diseases. Recently, the interest in delivery of proteins through the pulmonary route for treatment of lung diseases has significantly increased, especially after Covid-19 pandemic. The development of an inhalable protein combines the challenges of inhaled as well as biologic products since protein stability may be compromised during manufacture or delivery. For instance, spray drying is the most common technology for manufacture of inhalable biological particles, however, it imposes shear and thermal stresses which may cause protein unfolding and aggregation post drying. Therefore, protein aggregation should be evaluated for inhaled biologics as it could impact the safety and/or efficacy of the product. While there is extensive knowledge and regulatory guidance on acceptable limits of particles, which inherently include insoluble protein aggregates, in injectable proteins, there is no comparable knowledge for inhaled ones. Moreover, the poor correlation between in vitro setup for analytical testing and the in vivo lung environment limits the predictability of protein aggregation post inhalation. Thus, the purpose of this article is to highlight the major challenges facing the development of inhaled proteins compared to parenteral ones, and to share future thoughts to resolve them. Keywords Pulmonary delivery Proteins Dry powder for inhalation Aggregation Reconstitution Immunogenicity pmc-status-qastatus 0 pmc-status-live yes pmc-status-embargo no pmc-status-released yes pmc-prop-open-access yes pmc-prop-olf no pmc-prop-manuscript no pmc-prop-legally-suppressed no pmc-prop-has-pdf yes pmc-prop-has-supplement no pmc-prop-pdf-only no pmc-prop-suppress-copyright no pmc-prop-is-real-version no pmc-prop-is-scanned-article no pmc-prop-preprint no pmc-prop-in-epmc yes Introduction For decades, biologics therapies have shown tremendous success across different disease areas owing to their high target specificity and low toxicity. Currently, most biologics are formulated for parenteral delivery . The delivery of biologics by inhalation offers a non-invasive delivery route and higher local drug concentration compared to parenteral administration. 1 Pulmozyme®, a dornase alpha solution for nebulization by Genentech, has been in the market for 30 years for treatment of cystic fibrosis. 2 However, the failure of the first inhaled insulin product “Exubera” in the market, due to its bulky device and some adverse effects, 3 has decelerated the advancement of inhaled biological products. Afrezza®, an inhaled dry powder insulin by MannKind, is the only inhaled insulin in the market, yet its commercial performance is continuously challenged by the novel insulin delivery devices with minimum invasion and better dose accuracy. 2

, 4 Nearly 10 years after Exubera's withdrawal, there is a renewed interest in the development of inhaled biologics within the pharmaceutical industry. 5 Moreover, Covid-19 pandemic has put the inhalation route back in the spotlight for delivery of anti-covid drugs and/or vaccines. Currently, more than 60 biologic molecules are in different development stages in the pipelines. 6 As more inhaled biologic products are being developed, the innovators in this field are facing a lack of regulatory guidance regarding protein aggregation, stability or other characteristics that could impact the efficacy and/or safety for this novel route of administration. Thus, in this commentary, the authors aim to highlight the knowledge gaps in the inhaled biologics compared to parenteral ones, discuss challenges related to protein aggregation in formulations designed as dry powder for inhalation (DPI), and share some thoughts to potentially resolve them. Challenges Facing the Formulation of Proteins for Inhalation Biologics are mainly formulated as a solution for nebulization or DPI for pulmonary delivery. 7 Formulation of biologics as DPI is expected to have superior stability compared to solutions, however, the micronization and/or the drying steps impose major stability risks. 8

, 9 The biggest hurdle with different drying technologies is finding the optimum drying conditions to create respirable particles, with good aerosol performance without compromising the protein stability and product quality. Characterization of the physical properties of the dried powders, including size, size distribution, shape and porosity is a must to ensure successful pulmonary delivery. 10

, 11 For dry powder biologics for inhalation, it is equally important to evaluate the stability of the protein post drying, during storage and after actuation from device to ensure both efficacy and safety of the drug product. 1 Spray drying is the most commonly used technology for manufacturing inhalable dry powders. During spray-drying, the protein solution is sprayed into a preheated chamber to form dry particles which then get separated based on particle size and lastly collected. 10 Different biologics modalities including enzymes, peptides and monoclonal antibodies have been successfully spray-dried into dry powder. 8 Unlike lyophilization process, proteins are subjected to shear stress, air liquid interface and heat during spray-drying, all of which could significantly impact protein stability. 12 , 13 , 14 The prevention of protein denaturation during spray-drying requires fundamental understanding of the drying technology, the kinetics of droplet drying and the stability profile of the protein against the different stresses encountered during spray-drying. 15 Different excipients including sugars (trehalose, sorbitol), surfactants (polysorbate 20, polysorbate 80) and amino acids (lysine, histidine and arginine) have demonstrated protection of the protein during spray drying, and thus reduce protein aggregation. Protein stability in the dried powder could also be compromised by moisture exposure during storage, demanding low and controlled humidity conditions for proper storage. Lastly, possible interaction of the protein with the inhaler device should be assessed to exclude any drug-device incompatibilities. 16

, 17 Protein Aggregation in Parenteral Versus Inhaled Products The main inherent issue of protein instability is monomer loss and subsequent formation of aggregates. 18

, 19 Monomer loss potentially impacts the efficacy of the drug product while the formation of aggregates may cause an immunogenic response (for example, Inflammatory lung phenotype). Such response would be undesirable especially when the lung is the diseased organ being targeted for treatment and could potentially impose safety risks. 20

, 21 Currently, there is good understanding of the potential causes of protein instabilities leading to formation of aggregates of size varying from nm to µm and even as visible particles. 18

, 19 Over the years, the advancement of particle detection and characterization technologies in solutions together with better understanding around their safety aspects created the current guidelines for particle counts in parenteral solutions. 22 , 23 , 24 , 25 For sub-visible particles, there is the United States Pharmacopeia (USP) <788> guideline for parenteral products with limits of ≤ 6000/container and ≤ 600/container for sizes greater than or equal to 10µm and 25µm, respectively. 26 Such guidance allows for low level of particles, which inherently reduces risks of safety concerns caused by insoluble protein aggregates in the drug product. It is important to note that the guidance for sub-visible particles is not designed to de-risk protein aggregation in vivo or immunogenicity concerns, but rather to avoid the risk of capillary occlusion. 27 Pre-clinical in vitro models are increasingly employed to evaluate the protein stability post injection. 28 , 29 , 30 For instance, a novel protein-free serum in vitro setup revealed faster degradation profiles for two monoclonal antibodies (mabs) compared to accelerated stability studies. 30 However, the guidance for particulate levels serves as a unified regulation across parenteral formulations to limit particles, including protein aggregates, in the final product. Unlike parenteral products, there is no equivalent guidance with specifications applicable for protein particles in inhaled products. It is possible to employ light obscuration method of USP <788> for detection of particles, including protein aggregates in inhaled products, as a simple and robust approach for formulation and/or process screening purposes. For instance, the detection of higher levels of soluble and/or insoluble protein aggregates post nebulization or spray drying indicates that the formulation and/or the drying process led to protein unfolding and denaturation. 8

, 15 , 31 , 32 The analytical testing for protein aggregates should be designed to prevent creation or reduction of protein aggregation during sample collection 33 or handling. 27 Measurement of protein aggregation post reconstitution of bulk powder requires careful selection of the reconstitution media such as saline, buffer, simulated lung fluid, is required to avoid creating higher or lower levels of aggregates related to the reconstitution. Nevertheless, the challenge remains about setting specifications for such testing. Moreover, the level of protein aggregation detected in vitro is most likely different from the protein aggregation to occur, if any, in vivo post inhalation in the lungs. For example, inhaled dry powders are expected to disperse, deposit in the lung lining fluid, dissolve and then get absorbed. 34 Thus, the reconstitution of dry powder as bulk for analytical testing is not representative of the rehydration and dissolution of dry powder in the lungs, which may impose a different impact on the formation of soluble and insoluble protein aggregates. Risk of Protein Aggregation Post Inhalation The possibility and the extent of formation of protein aggregates in the lungs after inhalation of biologics aren't fully understood yet. The main function of the lungs is gas exchange, due to which the lungs are continuously in direct contact with the outside air. Thus, the lungs possess innate, cellular and humoral defense mechanisms to eradicate any foreign particulates and/or pathogens. 9 The upper airways are lined with thick mucus layer and beating cilia for mucociliary clearance, making biologics less efficacious in this region. The alveolar region provides a huge surface area with thin layer of lining fluid abundant with immune cells, such as alveolar macrophages, making it the main area for immunogenicity risks. 35 It is possible that any formed insoluble protein aggregates in the lungs would be rapidly cleared by alveolar macrophages. However, it is unknown if or when the tolerance threshold for such aggregates is broken down, resulting in exacerbation in immune response to such aggregates accumulating in the lungs. 36 Thus, the fate of the inhaled protein depends on its site of deposition along the respiratory tract, which is governed by the inhaled particle size, shape, density in addition to patient and inhaler device related factors. 37

The presence of protein aggregates in the lungs of animals within inhalation toxicology studies may be detected with light microscopy as eosinophillic materials in the airways, although only relatively large aggregates (≥10 µm) can be detected. To confirm the presence of the protein within the materials, more specific techniques, such as immunohistochemistry or Raman microscopy can be employed. The latter technique was used in a 4-week inhalation toxicology study in the rat with Serelaxin (a recombinant human relaxin-2), in which crystalline eosinophilic materials were observed within alveoli and/or bronchioles with light microscopy. Raman microscopy was used to confirm this material to be related to protein crystallization, most likely Serelaxin. It is of note that the findings were not present in a 4-week inhalation toxicology study with Serelaxin in the cynomolgus monkey indicating potential species differences. 38 Another study by Lasagna-Reeves et al, detected protein aggregates in the form of amyloid sheets in the mice lungs after inhalation of high doses of inhaled insulin. 39 Inhaled insulin products such as Exubera and Afrezza have reported increased number of insulin antibodies detected compared to subcutaneous route, but with no clinical adverse effects. 40 Such antibodies were linked in response to the molecule itself with no reports on protein aggregation in the lungs. 41 A recent study by Secher et al. reported immune-toxic events in the lungs of C57BL/6 mice after the pulmonary administration of IgG aggregates generated during nebulization of the antibody. 42 Lastly, a review article by Hall P et al., 43 which summarized the toxicology results of 12 different inhaled biologics, proposed adaptive immunity as the putative mechanism behind the most commonly observed lung pathology finding; perivascular/peribronchiolar mononuclear cell infiltrates and increased eosinophils in the bronchioalveolar lavage, although the potential for these findings to be exacerbated by protein aggregates is not known. Thus, it is rational to evaluate the potential for protein aggregation in the lungs as a precautionary approach based on the established correlation between protein aggregation and immunogenicity from parenteral products. 20

Mimicking the in vivo conditions encountered by the protein post administration, which may impact protein stability resulting in series of unfolding and aggregate formation (soluble and/or insoluble aggregates) remains challenging. In vitro dissolution studies for inhaled dry powders submerged in a large volume of medium in a dissolution cup or in a culture medium of lung cells can falsely alter the protein behavior in the drug product. Air-liquid interface (ALI) exposure of cells has been developed for better resemblance to in vivo conditions encountered by DPIs after deposition in the lung. 44 In one study by Leiske D. et al., protein unfolding at air liquid interface was detected by measuring the hydrophobicity for two mabs using Nile red fluorophore. 45 Currently, there are ALI culture models for different inflammatory lung diseases which could offer better correlation to the in vivo environment encountered by the protein post inhalation. 46 The local concentrations of the protein after dissolution can also affect the rate and extent of aggregate formation. 47

, 48 Unlike the lyophilized products, which are reconstituted to achieve a predefined concentration prior to administration 49 , a range of drug concentrations are expected along the respiratory tract due to its complex anatomy and based on the aerodynamic diameter of the drug particles which dictates the product deposition profile after inhalation. 50

, 51 The risk of protein aggregation in the lungs may be dependent on the inhaled dose, dosing frequency 52 and site of deposition, 35 which poses significant complexity on setting up the ALI culture studies to mimic the in vivo conditions. Conclusion and Future Thoughts Protein aggregation has been correlated with safety and immunogenicity concerns for parenteral products. 27

, 41 , 53 The FDA guidance for Immunogenicity Assessment for therapeutic proteins stated that inhalational route of administration is associated with increased immunogenicity compared to intramuscular and IV routes. 52 Thus, it is important to consider the risks of protein aggregation in the lungs post administration despite of not being routinely evaluated for parenterals. The immune response to protein aggregates post injection has long been studied 21

, 41 and clinically exhibited making the response detectable and measurable. 52 In contrast, the safety and immunogenicity risks for protein aggregation in the lungs is not fully understood yet. It is understandable that the early assessment for safety and immunogenicity of inhaled dry powder biologic relies on the preclinical studies. However, prediction of protein aggregation in lungs could potentially de-risk detection of immunogenic response in such studies, offering more confidence to proceed to clinical trials. Thus, all efforts are needed to lower risk of formation of protein aggregation in the lungs to reduce chances of immunogenicity. However, the question remains about a robust and reliable analytical protocol of rehydrating inhaled dry powder biologics to assess protein stability and potentially correlate with in vivo aggregation levels. With limited inhaled biologics approved on the market, the regulatory guidance for inhaled biologics needs to be elucidated further to clearly address challenges associated with developing inhaled biologics. Meanwhile, biopharmaceutical companies set their internal testing methods and specifications to ensure product safety and efficacy. Thus, to address these challenges, experts from academia, industry and regulatory sectors should collaborate to define the risk of protein aggregates in lungs and work towards a scientifically sound and unified guidance for assessment of protein aggregation in inhaled biologics. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References 1 Eedara BB Alabsi W Encinas-Basurto D Polt R Mansour HM. 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