A novel peptide excipient stabilizes DaxibotulinumtoxinA

Abstract

Surface adsorption of proteins in bulk drug product formulations and loss of protein structural integrity during manufacturing processes can diminish drug potency. In formulations containing low biologic drug substance, such as botulinum toxin type A (BoNTA) products, loss of active protein is a major concern. To mitigate the risk of activity loss, most commercial BoNTA products contain an abundance of human serum albumin (HSA, 125–1000 µg/vial) to prevent adsorption of BoNTA to container surfaces by competing for nonspecific binding sites. The stabilizing ability of HSA is well established; however, there are notable potential safety concerns surrounding its use as an excipient.

Our data suggests that at pH 5.5, in the presence of increasing concentrations of HSA the 150 kDa BoNTA core neurotoxin (RTT150) may form aggregate complexes with HSA when heated to 45–50 °C, in a dose-dependent manner. A new BoNTA product has recently been approved with a unique formulation which uses a proprietary synthetic cationic peptide (RTP004) to replace HSA as a stabilizer. Here, we show that RTP004 binds to the core neurotoxin and enhances its thermostability. In combination with polysorbate 20 (PS20), RTP004 efficiently and completely prevents surface adsorption of the 150 kDa core neurotoxin and stabilizes the biologic drug substance during manufacturing processes, even at concentrations 10- to 100-fold less than those used for HSA in commercial BoNTA products. Thus, the combination of RTP004 and PS20 forms the basis for a novel and effective BoNTA formulation, removing the need for HSA and thereby avoiding its theoretical safety risks and documented limitations.

Highlights

Introduction

Protein aggregation is a critical safety concern during production and delivery of biologic drugs, as aggregates potentially lead to immune responses or other side effects (Cromwell et al., 2006). Human serum albumin (HSA) is commonly included as a stabilizer/excipient in approved botulinum neurotoxins as well as other biotherapeutics to prevent biologic drug substance API protein aggregation and container surface adsorption (Velankar et al., 2024, Sattler et al., 2023). HSA-specific adverse events arising from use as a stabilizer/excipient have been identified as a risk, although the issue remains understudied (Sattler et al., 2023). The first concern is related to its source: most HSA used in commercial products is purified from human plasma. Although there are no known cases of transmission of blood-borne pathogens, there is always a risk of contamination (Hawe and Friess, 2007, López et al., 2004, Mead et al., 2007). A second concern is the potential formation of aggregates of HSA with the therapeutic protein, which may increase the propensity for development of neutralizing anti-drug antibodies. Such neutralizing antibodies may not only reduce or negate the efficacy of the therapeutics but may also impact endogenous proteins as well (Fradkin et al., 2009, Hermeling et al., 2004, Moore and Leppert, 1980, Rosenberg, 2006). For example, HSA aggregates in an interferon-α-2a protein therapeutic were linked to an increase in the immunogenic response against interferon-α-2a. The removal of HSA from the therapeutic reduced the number of patients with neutralizing antibodies targeting the therapeutic protein (Hochuli, 1997, Ryff, 1997).

Due to the risk of contamination and potential to elicit an immune response, there is movement away from use of HSA as a stabilizing excipient for protein therapeutics (Hawe and Friess, 2007, Jorgensen et al., 2009). The surfactant property of HSA can decrease the propensity of proteins to adsorb to container surfaces or aggregate at air–water interfaces by reducing the interfacial tension of the formulation (Khan et al., 2015, Lee et al., 2011). However, in modern formulations, nonionic surfactants including polysorbate 20 (PS20) can be used in place of HSA (Yi et al., 2020).

Although rare, neutralizing antibodies against BoNTA have been observed in patients treated with BoNTA products, in some cases leading to a loss in efficacy (Albrecht et al., 2019, Hefter et al., 2016, Müller et al., 2009, Yablon et al., 2005). As seen with other drug products containing HSA as an excipient (Cournoyer et al., 2004, Hochuli, 1997, Ryff, 1997), HSA aggregates may contribute as an adjuvant in the immune response to BoNTA.

DaxibotulinumtoxinA-lanm for injection (DAXXIFY®, Revance Therapeutics, Inc., Nashville, TN, USA), first approved by US Food and Drug Administration (FDA) in September 2022, comprises a 150-kDa type A botulinum toxin (RTT150) formulated in unique non-HSA excipients (Solish et al., 2021, Ellis et al., 2023). A key excipient is a proprietary, synthetic, 35-amino acid peptide (RTP004), intended to limit aggregation and prevent loss of BoNTA activity.

The sequence of RTP004 (RKKRRQRRRGKKKKKKKKKKKKKKKGRKKRRQRRR) consists of mainly basic amino acids that confer high positive charge to the peptide. Although RTP004 is unique in composition, other compounds have been investigated as potential stabilizers (Kudou et al., 2003, Sabbaghian et al., 2011) through shielding hydrophobic regions at near neutral pH (Fukuda et al., 2014). Here, we demonstrate that RTP004 in the presence of 0.05 % PS20 can replace HSA as an excipient and provide better thermostability for RTT150. We believe that the combination of RTP004 with PS20 in the formulation buffer containing histidine and trehalose at pH 5.5 will have an equal or better stabilization profile for RTT150 than that of HSA. PS20 prevents RTT150 adsorption on container walls as effectively as HSA while RTP004 interacts with RTT150 to stabilize the toxin and reduce thermally induced aggregation of the neurotoxin.

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Materials

The interaction between RTP004 and RTT150 was demonstrated using binding assays in a proprietary drug product formulation buffer (20 mM histidine and 18 % trehalose at pH 5.5, containing 0.05 % Polysorbate 20). Briefly, for binding assays, the surfaces of 96-well plates were precoated with RTP004 by incubating the excipient (in water) at concentrations ranging from 0 to 200 nM for 1 h at 37 °C. After incubation, unbound RTP004 was removed by aspiration, and the plates were rinsed five times with 0.05 % PS20 (Tween-20) in PBS (PBST). The plate was then blocked with ELISA blocking buffer (ThermoFisher, Catalog number N502) to minimize any nonspecific binding. Horseradish peroxidase (HRP)-conjugated RTT150 in formulation buffer (pH 5.5, containing 0.05 % PS20 and 1.8 mg/mL HSA) was added to each well at a single concentration of 0.5 µg/mL and the plates were incubated at 37 °C for 1 hr. For control comparison, an HRP conjugate of mouse antibody instead of RTT150 at 0.5 µg/mL was incubated with the coated RTP004 in the same buffer and plate.

Binding of RTT150 to custom monoclonal anti-BoNTA antibodies, Anti-BoNTA heavy chain or Anti-BoNTA light chain (both from Antibody Solutions), in the presence of RTP004 was assessed as follows: briefly, the surfaces of 96-well plates were precoated by incubating with the antibodies (in PBS) at 5 mg/mL for 1–7 days at ambient temperature. The coated plates were rinsed five times with 0.05 % PS20 (Tween-20) in PBS (PBST) and then blocked with ELISA blocking buffer (ThermoFisher, Catalog number N502). One hundred µL of RTT150 at 1.25 ng/mL (prepared by mixing one volume of 2.5 ng/mL RTT150 in formulation buffer with one volume of 50 mM Tris-HCl, pH 8.0, containing 1 % BSA and 0–160 µg/mL RTP004) were added to each well and the plate was incubated at 37 °C for 1 hr. After washing the plate, bound RTT150 was detected by incubation with a rabbit polyclonal antibody against BoNTA (Antibody Solutions) and subsequently anti-rabbit-HRP conjugate (Sigma, Catalog number SAB3700853) at ambient temperature.

Shaoqiu Zhuo, G. Reza Malmirchegini, Conor J. Gallagher, A novel peptide excipient stabilizes  DaxibotulinumtoxinA, International Journal of Pharmaceutics, 2025, 126238, ISSN 0378-5173, https://doi.org/10.1016/j.ijpharm.2025.126238.

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