Effects of buffers on spray-freeze-dried/lyophilized high concentration protein formulations

Abstract

Solid-state protein formulations are known to exhibit enhanced storage stability compared to their liquid dosage form counterparts. pH is one of the factors affecting the stability of protein formulations. The pH of protein formulations in the solution could be influenced by the buffer used, directly impacting their solid-state stability. During lyophilization, buffer components may interact with other formulation components present in the protein formulations, causing a pH shift. This study aimed to investigate the effects of phosphate buffer and amino acid buffers (such as histidine and/or arginine) on the physical properties and accelerated storage stability of spray freeze-dried or lyophilized protein formulations. A model protein, bovine serum albumin (BSA), was used to prepare high-concentration protein formulations. The formulations consisted of BSA, trehalose, and mannitol in an 80:15:5 ratio (w/w), respectively. Various buffers were utilized in the preparation of protein formulations, and the resultant solid formulations underwent screening via accelerated stability study using size exclusion chromatography (SEC). The combination of phosphate and arginine buffers resulted in increased monomer loss in the accelerated storage stability study. Additional characterizations, including solid-state Fourier transform infrared spectroscopy (ssFTIR) and powder X-ray diffraction (PXRD), were conducted. While these analyses did not definitively elucidate the mechanism behind the observed instability, their outcomes provide valuable insights for further investigation, highlighting the need for future research in this area.

Introduction

Biopharmaceutical research has been evolving after the 1950 s, including protein formulations, gene therapies, cell therapies etc. (Senior, 2023). Biopharmaceuticals are often made up of proteins with complex structures. These proteins in biopharmaceuticals are necessary for their functionality and are prone to degradation by several pathways like physical degradation involving aggregation or phase separation, and chemical degradation involving several hydrolytic and redox reactions (Mazzeo and Carpenter, 2009, Patel, 2011). Depending on the type of physical/chemical changes protein denaturation may be reversible or irreversible (Tanford, 1968), either of which may change the efficacy of the protein formulations.Fig. 1.Fig. 2.

Aggregation of protein is a type of physical instability where the single complex structure forms an oligomer, either through covalent or non-covalent bond formation (Zapadka, 2017). This aggregation process is due to interactions among the hydrophobic and hydrophilic functional groups present on the exterior or interior of the protein structures (Philo and Arakawa, 2009). The change in protein’s native state (partial unfolding) has also been observed as a driving force for aggregation process (Chi, 2003). Several factors such as change in temperature and pH, or stresses induced during manufacturing or storage can promote protein instability (Wang, 1999, Wang, 2005). These challenges can be addressed by formulating biologics with various stabilizing excipients such as buffers, cryoprotectants, bulking agents, surfactants, and other additives (Shire, 2009, Muralidhara and Wong, 2020).

Solid-state protein formulations are favored over their liquid counterparts due to their capacity to decrease molecular mobility and water activity, thereby extending the shelf life of proteins (Yoshioka and Aso, 2007, Miller et al., 2020). Various drying techniques are employed, such as lyophilization, spray drying, and spray freeze-drying (Ledet, 2015). Spray drying is a continuous heat assisted drying process which enables the formation of dry powder biologics (Ledet, 2015, Carrigy and Vehring, 2019). Lyophilization (or freeze-drying) and spray freeze-drying employ ultra-low temperatures and pressures to sublimate water to manufacture solid-state formulation (Sharma, 2021). Spray freeze-drying (SFD), a relatively novel technique, is particularly valuable for producing powders of temperature-sensitive biopharmaceuticals ensuring lesser heat stress on proteins than the spray drying process. In contrast to lyophilization, SFD enables the creation of spherical and porous particles with controlled sizes. During SFD, the liquid feed is sprayed into a cryogenic medium, facilitating rapid freezing of the protein formulation into its native state while preserving its structure. Subsequently, residual moisture is eliminated through the lyophilization process (Ledet, 2015). A notable advantage of SFD is the formation of highly porous spherical particles, which could enhance the powder flow properties (Lowe, 2018). These characteristics of SFD could be beneficial for drying high concentration protein formulations.

The term “High Concentration Protein Formulation” (HCPF) lacks a specific definition or regulatory guidelines from the Food and Drug Administration (FDA). It is noteworthy that HCPFs extend beyond the mere quantitative measurement of protein content, encompassing various attributes such as protein solubility, phase separation, and colloidal properties. Notably, a substantial portion of commercially available HCPFs comprises antibody products, typically falling within the range of 100–200 mg/ml (Wang et al., 2021). Protein concentration can alter the aggregation kinetics and nucleation, thereby impacting stability (Barnett, 2015). Some of the research studies suggested that aggregation in protein could be reduced at higher protein concentrations (Treuheit et al., 2002). This happens due to the increased resistance of protein towards denaturation and unfolding caused during processing (e.g., during freezing) (Chang et al., 1996, Carpenter, 1997). This was further known to be due to the reduction in protein-ice-water surface interaction (Carpenter, et al., 2002). While other study suggested that high protein concentration could cause an elevation in protein aggregation during lyophilization (Shire et al., 2004).

Solid-state high concentration protein formulations pose significant processing challenges such as high viscosity (15–25 cP) and longer reconstitution time (Salinas, 2010, Saluja, 2007, Neergaard, 2013, Cao, 2013, Kulkarni et al., 2020). The abundance of protein limits the inclusion of protective excipients, necessitating the use of multifunctional excipients. Amino acids such as histidine and arginine are commonly employed in FDA approved HCPFs due to their roles as buffering agents, viscosity reducers and protein stabilizers (Wang et al., 2021, Ren, 2023). Histidine has been shown to shield hydrophobic regions of the protein, whereas arginine increases the required free energy for initiation of protein aggregation (Saurabh, 2022, Shukla and Trout, 2010). Additionally, histidine and arginine have been demonstrated to reduce the viscosity of HCPFs in some instances. (Connolly, 2012, Inoue, 2014, Whitaker, 2017). Various combinations of buffers are utilized in FDA-approved HCPFs (Wang et al., 2021). Optimum pH is crucial for protein stability, as alterations can lead to partial or complete unfolding of the protein (Varga, 2016, Pyne et al., 2003). Selection of buffers is a crucial step as some buffers have been observed to crystallize during the freeze-drying process, potentially affecting overall stability by causing phase separation (Randolph, 1997, Shalaev, 2002). Additionally, some buffers can interact with stabilizing excipients such as sugars present in the formulation (Ohtake, 2004, Arsiccio and Pisano, 2018). Moreover, changes in buffer composition can influence changes in particle formation, thereby affecting surface area and overall stability (Mutukuri, 2023).

The aim of this study is to investigate the impact of amino acid buffers, potassium phosphate buffer, and their combinations on the stability of HCPFs. The potassium phosphate buffer was chosen over the sodium phosphate buffer, as sodium phosphate buffer tends to crystallize during freezing, while potassium phosphate buffer provides more stability for the protein upon lyophilization (Pyne et al., 2003, Pikal-Cleland and Carpenter, 2001, Thorat and Suryanarayanan, 2019). Commonly used amino acid buffers such as histidine and arginine, with chloride as the counter-ion were used. The rationale for using chloride counter-ion was that it has shown better compatibility with these amino acid buffers thereby enhancing protein stability (Stärtzel, 2015). Bovine serum albumin served as the model protein, while stabilizers like trehalose and mannitol were incorporated to protect the protein in the solid state and mitigate stresses during freezing and drying steps. Detailed formulation information is provided in Table 1. The formulations were prepared at a pH of 6.8 as the isoelectric point of BSA is in the range of 5.1–5.5 (Fologea, 2007). These formulations underwent drying via spray freeze drying (SFD) or lyophilization, and various physical characterizations including particle size and morphology, BET surface area, true density, and crystallinity were assessed. Furthermore, 90 days accelerated stability was conducted at 40 °C, with monomer loss analyzed using size exclusion chromatography (SEC). Changes in stability were investigated by assessing alterations in pH, secondary structure of the protein, and residual moisture content of the dried products.

Materials

Bovine serum albumin (BSA ≥ 98 %), trehalose dihydrate, D-mannitol, potassium phosphate monobasic monohydrate, potassium phosphate dibasic trihydrate, L-histidine monohydrochloride monohydrate, and L-arginine monohydrochloride were obtained from Millipore Sigma (St. Louis, MO, USA). 30 mL Slide-A-Lyzer™ Dialysis Cassettes, 10 K MWCO were obtained from Thermo Fischer Scientific (Waltham, MA, USA) for performing dialysis.

Chanakya D. Patil, Tarun Tejasvi Mutukuri, Kinnari Santosh Arte, Yijing Huang, Vinay Radhakrishnan, Qi Tony Zhou,
Effects of buffers on spray-freeze-dried/lyophilized high concentration protein formulations, International Journal of Pharmaceutics, 2024, 124974, ISSN 0378-5173, https://doi.org/10.1016/j.ijpharm.2024.124974.