On the role of excipients in biopharmaceuticals manufacture: Modelling-guided formulation identifies the protective effect of arginine hydrochloride excipient on spray-dried Olipudase alfa recombinant protein

Biopharmaceuticals are labile biomolecules that must be safeguarded to ensure the safety, quality, and efficacy of the product. Batch freeze-drying is an established means of manufacturing solid biopharmaceuticals but alternative technologies such as spray-drying may be more suitable for continuous manufacturing of inhalable biopharmaceuticals. Here we assessed the feasibility of spray-drying Olipudase alfa, a novel parenteral therapeutic enzyme, by evaluating some of its critical quality attributes (CQAs) in a range of excipients, namely, trehalose, arginine (Arg), and arginine hydrochloride (Arg-HCl) in the sucrose/methionine base formulation. The Arg-HCl excipient produced the best gain in CQAs of spray-dried Olipudase with a 63% reduction in reconstitution time and 83% reduction in the optical density of the solution. Molecular dynamics simulations revealed the atomic-scale mechanism of the protein–excipient interactions, substantiating the experimental results. The Arg-HCl effect was explained by the calculated thermal stability and structural order of the protein wherein Arg-HCl acted as a crowding agent to suppress protein aggregation and promote stabilization of Olipudase post-spray-drying. Therefore, by rational selection of appropriate excipients, our experimental and modelling dataset confirms spray-drying is a promising technology for the manufacture of Olipudase and demonstrates the potential to accelerate development of continuous manufacturing of parenteral biopharmaceuticals.

Introduction

Biopharmaceuticals provide new opportunities and challenges for manufacturing complex medicines that have massive potential to treat currently incurable diseases. For injectables, aqueous biopharmaceutical formulations are preferred but protein–water interactions can destabilize the protein, triggering biochemical degradation pathways such as hydrolysis, deamination, oxidation and aggregation of proteins (Carpenter et al., 2002). Hence, the controlled removal of water confers important benefits including improved stability, prolonged shelf-life, ease of storage, and reduced cost of transportation at ambient temperatures (Langford et al., 2017, Moeller and Jorgensen, 2008, Shalaev et al., 2008, Tang and Pikal, 2004b). To date, freeze-drying is the gold standard for manufacturing biopharmaceuticals but drawbacks include high operational and maintenance costs, reduced efficiency, challenges with respect to heat and mass transfer, scale-up and inability to produce free-flowing powder (Salnikova et al., 2015,Tang and Pikal, 2004a, Tang and Pikal, 2004b). In order to overcome the disadvantages associated with conventional shelf-based freeze drying, alternative drying technologies must be evaluated to develop safe and cost-effective ways to manufacture biologics (Sharma et al., 2021). Spray-drying is a promising candidate for the biopharmaceutical industry as it is cost effective, continuous, and improves production efficiency (Roser, 1991,Santivarangkna et al., 2007,Walters et al, 2014). This technique is particularly suited to thermolabile products as the evaporation process is very fast (taking from milliseconds to a few seconds) thereby minimizing exposure to high temperatures (Celik and Wendell, 2010). The production of millions of small droplets provides a large surface area for heat and mass transfer allowing rapid evaporation. However, factors such as uncontrolled dehydration, large increases in temperature, shear and protein adsorption at interfaces have been reported to affect the stability of proteins (Rajan et al., 2021).

To protect and preserve the stability of proteins during freeze-drying and spray-drying, a wide range of excipients such as amorphous saccharides, polyols, amino acids and surfactants have been explored (Narhi et al., 2012) . Spray drying technology has recently been successfully applied to antibody-based formulations (Duran et al., 2021, Fiedler et al., 2021, Massant et al., 2020, Pan et al., 2022, Shepard et al., 2022, Shepard et al., 2021, Tejasvi Mutukuri et al., 2021). Four main mechanisms have been reported that partly explain the stabilizing effect of different excipients during protein dehydration (Ajmera and Scherließ, 2014, Elversson and Millqvist-Fureby, 2005). The first proposed mechanism is the “water replacement hypothesis” which states that stabilizers such as sucrose and trehalose form hydrogen bonds at specific sites on the surface of the protein during dehydration. As water is removed from the protein solution, the hydrogen bonds between the protein and water molecules are disrupted. The hydrogen bonds formed between the stabilizer and protein create a water-like environment that stabilizes the native structure of the protein (Allison et al., 1999, Carpenter et al., 1994, Kreilgaard et al., 1999, Starciuc et al., 2020). Protein–protein interactions are decreased upon addition of sugars, reducing protein aggregation (Costantino et al., 1994). The alternative “glass dynamics mechanism” proposes that amorphous stabilizers such as sucrose, trehalose, glucose and raffinose form a rigid, inert matrix around protein molecules wherein the motion of the protein is coupled to the motion of the matrix, thereby limiting its structural relaxation and preserving the tertiary structure (Chang et al., 2005, Green and Angell, 1989, Ying et al., 2012, Zhao et al., 2018). By contrast, the theory of “preferential exclusion/interaction” states that excipients such as sucrose, trehalose, mannitol and sorbitol in solution are preferentially excluded from the surface of proteins, thereby stabilizing the native structure of the protein as a result of increased chemical potential and interaction with water molecules (Arakawa and Timasheff, 1982, Carpenter et al., 1994, Lerbret et al., 2008, Sudrik et al., 2019). The final alternative is the “reducing surface adsorption hypothesis” which states that protein adsorption at the surface of the drying layer is reduced in the presence of surfactants such as polysorbate 20 and polysorbate 80, thereby preventing protein denaturation at the air–liquid interface (Arsiccio and Pisano, 2018, Maa et al., 1998).

In the present study, we characterise the stability of a spray-dried enzyme-based formulation and compare our measurements with atomic-resolution models generated from molecular dynamics (MD) computer simulations. By mapping between the experimental measurements and the model predictions, we identify the protein–excipient interaction networks created with various excipients. The enzyme formulation we study is Olipudase alfa (Sanofi, , 2022, Zhou et al., 2016). Sold under the brand name XenpozymeTM, it is a therapeutic sucrose-based formulated drug substance (FDA, 2022). The Active Pharmaceutical Ingredient (API) is a copy of the normal acid sphingomyelinase enzyme, and is the first disease-modifying treatment of non-central nervous system manifestations of acid sphingomyelinase deficiency (ASMD) type A/B or type B. Also known as acid sphingomyelinase-deficient Niemann Pick disease, ASMD is a rare progressive genetic disorder linked to deficiency of the enzyme acid sphingomyelinase and hence inability to metabolise the sphingomyelin lipid. Here we evaluate the potential protective effect of sucrose, trehalose, arginine and arginine hydrochloride excipients, which are among the most commonly used stabilizers in freeze-dried and spray-dried protein formulations (Sharma et al., 2021) yet little is known about their mechanisms of interaction with proteins during freeze-drying and spray-drying (Bjelošević et al., 2020, Pinto et al., 2021). Here, the critical quality attributes (CQAs) of Olipudase were evaluated in terms of its reconstitution properties as measured by UV–Vis spectroscopy and Size Exclusion Chromatography (SEC) and supported by atomically-detailed MD simulations of the protein–excipient interface in aqueous solution. To the best of our knowledge, the stability of spray dried Olipudase has not been studied before. Here we resolve its protein–excipient interaction networks and demonstrate the importance of selecting the most appropriate formulation components in seeking to protect fragile biologics during processing.

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Materials

Trehalose, L-arginine (Arg) and L-arginine hydrochloride (Arg-HCl) were purchased from Sigma Aldrich, Ireland and used without further purification. Formulated liquid and lyophilized Olipudase with 5 % (w/v) sucrose and 100 mM methionine in sodium phosphate buffer at pH 6.5 was used as received from Sanofi, Waterford, Ireland. 5 % (w/v) each of trehalose, Arg and Arg-HCl were separately added to make the alternative formulations.

Ashutosh Sharma, Pierre Cazade, Ambrose Hayden, Dikshitkumar Khamar, Damien Thompson, Helen Hughes, On the role of excipients in biopharmaceuticals manufacture: Modelling-guided formulation identifies the protective effect of arginine hydrochloride excipient on spray-dried Olipudase alfa recombinant protein, International Journal of Pharmaceutics, 2024, 124466, ISSN 0378-5173, https://doi.org/10.1016/j.ijpharm.2024.124466.

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