Optimizing high-drug-loaded spray-dried antibody fragment formulations for sustained delivery: influence of excipient composition and humidity on long-term stability

Abstract

To facilitate the development of protein depots for sustained delivery, this study optimized spray-dried formulations of a model antibody fragment (Fab2) by evaluating the impact of excipient composition and environmental stress on long-term stability. Sucrose and trehalose were compared at low excipient-to-protein mass ratios (0.3:1 and 0.5:1), alongside the effects of polysorbate 20 and sodium chloride, under storage conditions mimicking physiological temperature (37 °C) at both low (sealed vials) and high (open vials) 80% relative humidity. While trehalose formulations exhibited significantly higher glass transition temperatures than sucrose-based systems, both disaccharides provided comparable protection against aggregation. Exposure to high humidity accelerated degradation across all formulations, resulting in massive particle growth, increased aggregation, and the formation of basic chemical variants including pyroglutamate and succinimide species. These findings underscore that while spray drying can engineer suitable particles for delivery, ensuring therapeutic viability requires maintaining specific excipient thresholds and strictly controlling moisture ingress.

Introduction

Protein therapeutics, including antibody fragments (Fabs), have revolutionized the treatment of chronic diseases. However, their intrinsic physical and chemical instability in the liquid state poses significant challenges for manufacturing and storage. To overcome these limitations, solid-state processing methods such as lyophilization and spray drying are commonly employed (Manning et al. 2010). By converting proteins into an amorphous solid matrix, typically in the presence of stabilizing disaccharides, molecular mobility is restricted, thereby preserving protein structure and activity (Prestrelski et al. 1993; Jang et al. 1995; Sollohub and Cal 2010).

While lyophilization is the standard for shelf-stable vials, spray drying offers distinct advantages for advanced drug delivery systems. Unlike the irregular cakes produced by freeze-drying, spray drying enables the engineering of micron-sized, spherical particles with controlled density and morphology (Sollohub and Cal 2010). These attributes are critical for drug delivery and developing sustained-release depots, such as poly(lactic-co-glycolic acid) (PLGA) solvent-depot, high-concentration suspensions, microspheres or implants, where particle characteristics directly influence drug encapsulation efficiency and release kinetics (Andya et al. 1999; Chang et al. 2015).

A major constraint in developing protein depots is the requirement for high drug loading to minimize the implant size and reduce dosing frequency. Conventional solid-state formulations generated via drying techniques, including spray drying and lyophilization typically utilize high excipient-to-protein ratios, ranging from 1:1 to 10:1 (w/w), to ensure robust stabilization. However, such high excipient loads dilute the active ingredient, rendering them unsuitable for volume-constrained depot applications where prolonged drug release is desired. Consequently, there is a critical need to evaluate protein stability at substantially reduced excipient ratios (e.g., 0.3:1 or 0.5:1 w/w), a regime where the protective capacity of the sugar matrix may be compromised. Maximizing drug loading is important to us for the purpose of long-term drug delivery without frequent dosing. Consequently, our investigation has primarily focused on the stability performance of a model protein at relatively low excipient-to-protein mass ratios (0.3:1 or 0.5:1 w/w sugar to protein), which are substantially lower than the conventionally reported ratios in the literature, typically ranging from 1:1 to 10:1 (w/w) (Cleland et al. 2001; Rajagopal et al. 2019). Although earlier reports have included formulations with 0.3:1 and 0.5:1 excipient‑to‑protein ratios, they generally did so within a wider set of conditions and did not systematically evaluate stability at these low ratios. Our work addresses this gap by focusing directly on performance under these minimal excipient conditions and high drug‑loading scenarios.

Furthermore, once encapsulated within a polymer depot, the protein is exposed to a harsh physiological environment characterized by body temperature (37 °C) and increasing humidity as the polymer matrix hydrates and degrades. Understanding the interplay between relative humidity (RH), excipient composition, and protein stability is essential for predicting performance in vivo.

In this study, we employed spray drying to develop high-drug-loaded solid-state formulations of a model antibody fragment (Fab2). Spray drying is advantageous for protein depot systems due to rapid particle formation, tunable particle size/morphology critical for drug release kinetics and compatibility with high drug loading (Wan and Yang 2016; Butreddy et al. 2021; Pinto et al. 2021; Michaelides et al. 2024). We systematically evaluated the influence of excipient composition on protein stability, specifically comparing two disaccharides (sucrose and trehalose) at challenging, low excipient-to-protein ratios (0.3:1 and 0.5:1 w/w) (Fig. 1). Additionally, the impact of secondary additives, including a surfactant (polysorbate 20) and salt (sodium chloride), were examined as formulation variables to assess how interfacial protection and ionic strength influence stability within the spray‑dried solid‑state matrix.

Stability was assessed under conditions mimicking the depot environment: 37 °C storage under both low moisture (sealed) and high moisture (80% RH) conditions. Studies performed at 37 ℃ revealed that the appropriate molar ratio of sugar to protein stabilized Fab2 against aggregation, preserving its antigen binding capacity for up to 3 months. Furthermore, under high humidity conditions, the presence of trehalose or sucrose at an excipient-to-protein molar ratio of 70:1 (i.e. 0.5 w/w) resulted in reduced Fab2 instability due to protein aggregation, compared to lower excipient ratios or formulations lacking excipients altogether. Therefore, our study findings are suggestive of compatibility with depot-like environments where long-term drug storage inside a PLGA solvent depot formulation at physiological conditions can be achievable even for high concentration protein formulations. Overall, a specific sugar/protein molar ratio of even 35 to 1 (i.e. 0.3 w/w) was sufficient to improve the physical stability of spray-dried Fab2 up to 3 months where storage humidity was lower. By characterizing aggregation, chemical degradation, and antigen-binding capacity, this work aims to identify the formulation boundaries required to balance maximal drug loading with long-term therapeutic viability. In particular, the observed effects of moisture content, excipient composition, and processing conditions on protein stability are equally relevant and broadly applicable for spray-dried formulations intended for high-concentration polymeric drug delivery systems, where maintaining solid-state integrity and enabling efficient redispersion are critical (Marschall et al. 2023; Jons et al. 2025).

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Materials

The model antibody fragment (Fab2) was obtained from Genentech (South San Francisco, CA) and is the same fragment antibody reported in previous studies (Nayak et al. 2020). Sucrose and α,α-trehalose dihydrate were purchased from Ferro Pfanstiehl Laboratories (Cleveland, OH), and sodium chloride (NaCl) was obtained from J.T. Baker (Phillipsburg, NJ). L-Histidine monohydrochloride (Histidine-HCl) was purchased from Sigma-Aldrich (St. Louis, MO), and polysorbate 20 (PS20) was obtained from Spectrum Chemical (New Brunswick, NJ). All other chemicals were of analytical grade and used as received.

Nayak, P., Rajagopal, K. & Chang, D. Optimizing high-drug-loaded spray-dried antibody fragment formulations for sustained delivery: influence of excipient composition and humidity on long-term stability. AAPS Open 12, 44 (2026). https://doi.org/10.1186/s41120-026-00182-2

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