Monoacyl phospholipids to replace polysorbates as interfacial stabilizers in parenteral monoclonal antibody formulations

Protein-based drugs are prone to both physical and chemical instability in aqueous solutions. Surfactants, such as polysorbates (PS), are commonly employed to mitigate interfacial stress, thus preventing protein aggregation and particle formation. However, polysorbates can undergo enzymatic hydrolysis by residual host cell proteins and oxidation during long-term storage in parenteral formulations. This can lead to the generation of free fatty acid particles, inadequate protein stabilization, and protein oxidation. In this study, we investigated several monoacyl phospholipids (MAPLs) with varying fatty acid chains as potential alternative surfactants for monoclonal antibody (mAb) formulations and compared their efficacy to the industry standard, polysorbate 80.

The hemolytic activity of MAPLs was tested using erythrocytes in 95 % plasma. All MAPLs prevented mAb particle formation during shaking and freezing-thawing at surfactant concentrations several orders of magnitude below the threshold for hemolysis, suggesting that the risk of erythrocyte damage from MAPLs is non-critical. Stabilization of mAbs occurred around the critical micelle concentration, which were comparable to that of PS80, but MAPLs achieved lower interfacial tension values.

MAPLs were found to be more resistant to enzymatic hydrolysis by porcine liver esterase and forced oxidation than PS80. After long-term liquid storage, lyso-myristoyl-phosphatidylcholine (LPC 14:0) at low concentrations provided superior mAb stabilization to PS80, which exhibited substantial chemical degradation. At higher concentrations, both PS80 and LPC 14:0 showed a decrease in surfactant concentration. Lyophilization enhanced mAb stabilization relative to liquid formulations, with MAPLs performing as well as PS80 at high concentrations and outperforming PS80 at low concentrations. MAPLs also better preserve the siliconization in pre-filled syringe (PFS) barrels compared to PS80. In short, MAPLs demonstrate mAb stabilization and chemical stability comparable to, and in some cases superior to, PS80, making them a promising alternative as interfacial stabilizers in parenteral protein formulations and warranting further exploration.

Introduction

Therapeutic proteins have become essential components of modern medicine, particularly with the rise of monoclonal antibodies (mAbs). Over half of the biopharmaceuticals approved between 2019 and 2022 were mAbs, highlighting their critical role in treating a range of diseases, including cancers, autoimmune disorders, and infections diseases (Ghosh et al., 2023; Kothari et al., 2024). Despite their success, protein drugs face several challenges throughout their lifecycle that compromise their chemical and physical stability. A major contributor to this instability is their interaction with and adsorption onto various interfaces, due to their amphiphilic nature. Protein adsorption can lead to the formation of interfacial films and partial unfolding of the protein structure (Deiringer and Friess, 2022; Ghazvini et al., 2016; Koepf et al., 2018; Rudiuk et al., 2012). Rupture of these films can cause aggregates, in many cases as particles, formed at the interface and released into solution, negatively impacting not only the quality of the drug, but also its safety and efficacy.

Many protein drugs are stored frozen or lyophilized to enhance long-term stability. In these states, the mobility of molecules is reduced, which limits chemical reactions, microbial growth, and protein self-interactions. Nevertheless, protein aggregation can occur at the ice-liquid interface during these processes (Bluemel et al., 2022a, 2022b; Chang et al., 1996). While the mechanism of particle formation is not yet fully understood, theories such as protein cold denaturation have been proposed (Lazar et al., 2010; Seifert and Friess, 2020).

Additionally, protein formulations can be stored in pre-filled syringes (PFS), which offer reduced risk of contamination, more precise dosing, and enhanced patient convenience. Syringe barrels are typically coated with silicone oil (SO) for lubrication, which ensures ease of administration and patient comfort. Similar to their behavior in other containers, proteins can adsorb to the SO coating, which again can lead to protein aggregation. SO can migrate into the bulk solution, providing more surface area available for protein adsorption. While surfactants can help prevent protein interaction with SO, they can also promote the migration of SO droplets into solutions. Moll et al. found that the inclusion of PS20 in formulations facilitated silicone depletion, with different mAb molecules also affecting SO migration. They suggested a correlation between interfacial tension and the stability of the SO layer (Moll et al., 2024). Several studies have found that proteins are destabilized by the formation of silicone droplets and mixed SO-protein aggregates increase the risk of immunogenicity in patients (Chisholm et al., 2017; Gerhardt et al., 2014; Jones et al., 2005; Kannan et al., 2021).

To mitigate protein adsorption, surfactants are commonly used as excipients. These small amphiphilic molecules can diffuse and adsorb quickly, preventing protein adsorption and inhibiting the formation of protein films. Surfactants can also occupy the ice-liquid interface, protecting protein molecules from freezing or drying stress. Even though certain surfactant-protein combinations can interact and impede protein self-interaction or adsorption, the excipients in this study stabilize proteins via competitive adsorption (Papadopoulos et al., 2024). Polysorbates (PS) 20 and 80 are used in approximately 90 % of the surfactant-containing parenteral protein formulations. Other approved surfactants include poloxamer 188 or 171, though these are considered less effective for protein stabilization (Gervasi et al., 2018; Grapentin et al., 2020; Soeda et al., 2023).

Polysorbates are prone to hydrolysis and oxidation during long-term storage, which can lead to the generation of undesirable byproducts that may further promote protein or free fatty acid particle formation and protein degradation (Larson et al., 2020; Roy et al., 2021; Weber et al., 2023). Surveys indicate that approximately two-thirds of companies observed PS hydrolysis, oxidation, or both in at least one of their drug products (Wuchner et al., 2022a). Literature suggests that impurities in protein solutions, originating from the host cells, substantially contribute to PS degradation via enzymatic hydrolysis. Carboxylesterases, along with other enzymes such as lipases and phospholipases, have been identified as key culprits in these processes. Highly concentrated protein solutions with lower PS content are particularly vulnerable to these issues. In such cases, host cell proteins (HCPs) are present in excess compared to PS, which intensifies the degradation effect (Wuchner et al., 2022b). Additionally, oxidation in PS solutions, due to auto-oxidation or driven by the presence of peroxides, can catalyze protein oxidation (Kishore et al., 2011; Kranz et al., 2019; Liu et al., 2022). This effect has even been observed in lyophilizates, although to a lesser extent than in liquid formulations. Furthermore, PSs are associated with hypersensitivity and anaphylactic reactions, further complicating their use in therapeutic protein formulations (Coors et al., 2005; Schwartzberg and Navari, 2018).

Monoacyl phospholipids (MAPLs), also known as lysophospholipids, present a promising alternative to the established, yet far from ideal, surfactants. These are monoacyl components found in natural lecithins and can be enzymatically derived from natural sources such as soybean or egg, or synthesized at the sn-1 position (Drescher and Hoogevest, 2020). MAPLs present enhanced micellar solubility and reduced susceptibility to hydrolysis compared to diacyl phospholipids. MAPLs are used in enzyme-modified lecithin for oral administration, as food additives, in self-emulsifying drug delivery systems, and in mixed micelle formulations. MAPLs are physiological constituents found in bile salts and in the blood, and they are recognized as safe (GRAS) by the US FDA (Hoogevest et al., 2011). Furthermore, the use of MAPLs as excipients in solid dispersions, nanostructured lipid carriers, and as treatments for metastatic tumor cells has been studied (Gautschi et al., 2015; Raynor et al., 2015; Wolf et al., 2018).

Though diacyl phospholipids are frequently used in parenteral formulations, such as oil-in-water emulsions, liposomes, and mixed micelle systems, the hemolytic activity of MAPLs has prevented their use in parenteralia (Hoogevest et al., 2011; Reman et al., 1969; Weltzien, 1973). However, a recent study demonstrated that hemolysis by MAPLs in serum is much less critical than previously thought. The concentration of S LPC 80 needed to cause 50 % hemolysis (HC50) increased 65-fold when tested in plasma versus buffer due to the binding of S LPC 80 to albumin (Papadopoulos et al., 2024). MAPLs used in this study were similarly tested for hemolysis, using a low concentration of erythrocytes diluted in plasma. This technique was used previously (Papadopoulos et al., 2024), and was adapted from a similar method used by (Hartl et al., 2023). Where traditionally hemolysis assays are performed with washed erythrocytes in buffer, with red blood cell concentrations of 1–4 % (Sæbø et al., 2023), this procedure used unwashed erythrocytes diluted in plasma to retain the stabilizing effects of albumin.

Recently, MAPLs have been shown to effectively prohibit mAb aggregation caused by shaking stress or peristaltic pumping by means of competitive adsorption (Papadopoulos et al., 2024). This study explores several additional MAPLs with varying fatty acid chains and compares their efficacy to the industry standard polysorbate 80.
Proteins can adsorb and aggregate when exposed to various interfaces during manufacturing, transport, and application. To investigate this, we subjected mAb formulations to mechanical and freezing stress. The combination of the critical micelle concentration (CMC) and equilibrium surface tension was analyzed in relation to the protein-stabilizing ability of the surfactants.

Polysorbates are particularly problematic due to their chemical instability. Therefore, surfactants were subjected to forced hydrolysis using a model for HCPs and forced oxidation. Additionally, both liquid and lyophilized mAb formulations containing select surfactants were stored long-term at several temperatures to simulate product shelf life. Samples were assessed for protein and surfactant stability. Surfactant-mAb formulations were also stored in siliconized syringes and evaluated for protein aggregation, syringe functionality, and the stability of the SO coating.

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