A thermodynamic investigation into protein–excipient interactions involving different grades of polysorbate 20 and 80

Developing stable biopharmaceutical formulations is of paramount importance and is typically achieved by incorporating surfactants as stabilising agents, such as polysorbate 20 and 80. However, little is known about the effect surfactant grade has on formulation stability. This study evaluates the effect of regular grade and Super-refined™ polysorbates 20 and 80 and their interaction with model proteins, namely β-lactoglobulin (β-Ig), human serum albumin (HSA) and immunoglobulin gamma (IgG), using isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). ITC results indicated that all four polysorbates underwent binding interactions with β-Ig and HSA, yet no interaction was observed with IgG this is postulated to be a consequence of differences in secondary structure composition. Surfactant binding to β-Ig occurred at ratios of ~ 3:2 regardless of the surfactant used with dissociation constants ranging from 284 to 388 µM, whereas HSA bound at ratios of ~ 3:1 and dissociation constants ranging from 429 to 653 µM. Changes in enthalpy were larger for the surfactant interactions with HSA compared with β-Ig implying the former produced a greater binding interaction than the latter. DSC facilitated measurement of the temperature of unfolding of each protein with the presence of each polysorbate where results further confirmed interactions had occurred for β-Ig and HSA with an increased unfolding temperature between 4 and 6 K implying improved protein stability, yet again, no interaction was observed with IgG. This study thermodynamically characterised the role of polysorbates in protein stabilisation for biopharmaceutical formulations.

Introduction

Biopharmaceuticals, also known as biologics, are becoming the most effective method for handling conventionally difficult to treat diseases such as cancer and Alzheimer’s disease [1]. These drugs are usually delivered to patients using parental routes of administration [2], with monoclonal antibodies (mAbs) being the most frequently used system [3]. However, it has been well documented that biologics are incredibly susceptible to degradation via several routes including chemical changes to constituent amino acids, as well as physical changes to proteins in the form of aggregation and unfolding [4,5,6]. Physical degradation is a common problem in the formulation stage of biologic development and tends to result in the formation of oligomers [7]. This occurs via several different mechanisms including reversible oligomerisation of native proteins [8], aggregation due to chemical changes [9] and adsorption of proteins on to the surfaces of containers causing changes in protein structure which leads to aggregation [7]. Both reversible oligomerisation and irreversible oligomerisation, in addition to surface-induced aggregation, are problems that can occur regardless of the stability of the protein molecules themselves. Overcoming these susceptibilities to produce a stable formulation can be achieved by adding excipients such as surfactants [10]. The most common surfactants in use are polysorbates 20 and 80 (Fig. 1), available as Tween™ 20 and Tween™ 80 by Croda Ltd. [11]. Polysorbate 20 and polysorbate 80 are used due to their non-ionic nature and low toxicity [12]. Their structure consists of a sorbitan molecule that has undergone esterification with polyoxyethylene (POE) and then further esterification of the POE chains with lauric acid for polysorbate 20 and oleic acid for polysorbate 80 [13, 14].
It has been shown that polysorbate 20 and polysorbate 80 are often included in biologic formulations at concentrations of around 0.11 mM and 0.19 mM, respectively, ten times lower than the critical micellar concentration (CMC) reported in a recent study [15]. Surfactants stabilise biologic formulations against surface-induced aggregation via displacement of proteins at interfaces by preferential competitive adsorption [16, 17]. In order to stabilise against reversible oligomerisation of native proteins, surfactant molecules interrupt bonding interactions of proteins; this type of interaction has been investigated using various different proteins as model biologics, including bovine serum albumin (BSA) and different surfactants, such as dodecyltrimethylammonium bromide (DTAB) [18,19,20]. Investigations into proteins and surfactant protein interactions are often conducted using differential scanning calorimetry (DSC) [21,22,23,24,25,26] and isothermal titration calorimetry (ITC) [18, 27]. Garidel et al. have investigated the interactions of human serum albumin and immunoglobulins with polysorbates 20 and 80 using ITC and DSC; they concluded that HSA interacted with polysorbates 20 and 80 with an association constant of approximately 103 M−1 and a protein to polysorbate stoichiometry of 1:3. They also found that IgG did not interact with either polysorbate [28] for the one polysorbate grade studied. These two particular calorimetric techniques are often used for such studies for several reasons, firstly they are label-free meaning that the samples can remain unaltered for a more representative experiment, secondly they allow the determination of multiple parameters simultaneously. For example, DSC can produce results identifying changes in heat capacity and the thermal stability of a sample which are often used to infer the denaturation point of a protein, as well as changes in total enthalpy and Van’t Hoffs enthalpy leading to the determination of changes in Gibb’s free energy [29, 30].

Polysorbate 20 and 80 are commercially available at several levels of purity, known as grades, including regular grade (RG) and Super-refined™ (SR). Being of a higher grade, SR polysorbates differ from their RG counterparts with respect to their impurity tolerance, for example they contain a reduced peroxide content [31]. Few studies have compared different purities of polysorbate 20 and polysorbate 80 and their effects on the stability of biologics. One study by N. Doshi et al. investigated the effects of oxidative and reductive degradation of polysorbate 20 using two distinct purities, high purity (HP) and SR through degradation by enzyme interactions [32]. They concluded that SR polysorbate 20 was more prone to degradation than HP polysorbate 20 due to the increased oleate ester content and this led to an increased risk of aggregation.

This study considers two grades of polysorbate 20 and 80 to consider the effect of different polysorbates and grades on their interactions along with three model proteins ranging in size from 16 to 157 kDa to investigate whether surfactant interactions are protein-specific and/or polysorbate specific. Using ITC and DSC to characterise interactions utilising stoichiometry, binding constants, enthalpy changes and thermal stability will help formulators further understand how surfactants stabilise proteins, the nature of such interactions and ensure the most suitable surfactant is selected for each new biopharmaceutical formulation.

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Materials

Polyoxyethylene (20) sorbitan monolaurate (‘polysorbate 20’) and polyoxyethylene (20) sorbitan monooleate (‘polysorbate 80’) were donated by Croda Europe Ltd.: standard compendial grades, referred to as Tween™ 20 (BN:50,702) and Tween™ 80 (BN:49659A), and high purity grades referred to as Super Refined™ Polysorbate 20 (BN:0001814116) and Super Refined™ Polysorbate 80 (BN:0001779440). The Super Refined versions are distinct from the standard grades through their chemical composition including a low peroxide value (2.0 meq O₂/Kg max.), limited formaldehyde (10 ppm max.), low residual EO (1 ppm max.), low 1,4-dioxane (5 ppm max.), low residual Na and K (5 ppm max.), low moisture (0.2% max.), decreased cellular irritation and microbial testing [31]. Three proteins were supplied by Sigma-Aldrich, UK, as lyophilised powders: immunoglobulin G (IgG) from human blood (> 99%), albumin from human serum (HSA) (> 96%) and β-lactoglobulin (β-Ig) from bovine milk (> 90%). These proteins were chosen to cover a range of molecular sizes from IgG (155 kDa) to HSA (65 kDa) to β-Ig (18 kDa). Potassium phosphate saline buffer of pH 7.4 was composed of 1.8 mM KH₂PO₄ (> 99%, Sigma-Aldrich, UK), 8.2 mM K₂HPO₄ (> 98%, Sigma-Aldrich, UK), 2.7 mM KCl (> 99%, Fisher Scientific, UK),140 mM NaCl (99.5%, Acros Organics, UK) and ultra-pure water (18.2 MΩ·cm).

Whiteley, J., Waters, L.J., Humphrey, J. et al. A thermodynamic investigation into protein–excipient interactions involving different grades of polysorbate 20 and 80. J Therm Anal Calorim (2024). https://doi.org/10.1007/s10973-024-13533-6

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CPHI Milan 2024 – with a focus on excipients