(T1030-08-54) Evaluation of Glycerol and Sorbitol in Preventing Aggregation of Monoclonal Antibodies under Mechanical Stress

Purpose: Biopharmaceuticals led by monoclonal antibodies (mAbs) have become the fastest growing class of drugs in the market in the past two decades. However, due to the physical and chemical instability of proteins, the development and production of biologic products remain challenging. It has been reported that aggregates of proteins tend to increase immunogenicity, seriously affecting the safety and efficacy of the products[1]. Among different stresses that cause the aggregation of therapeutic proteins, mechanical and interfacial stresses are commonly experienced by products and agitation methods in the form of shaking and stirring of protein solutions have been used to assess the susceptibility of mAbs to aggregation. Optimization of formulation is one of the ways in which drug developers improve the stability of biological drugs against these stresses. The addition of excipients such as carbohydrates, polyols, amino acids and surfactants is widely used to stabilize proteins. Currently the most commonly used surfactants in biologic products are polysorbate 80 (PS80) and polysorbate 20 (PS20) with known mechanisms for surfactant induced stabilization[2]. On the other hand, polysorbates are known to degrade during long-term stability, leading to the exploration of novel excipients. Structurally, some polyols with hydrophobic carbon chains and hydrophilic hydroxyl groups at different sides of the molecule exhibit amphiphilic features, suggesting a possible interfacial activity that could prevent the need for surfactants[3]. In line with this hypothesis there are products in the market that contain glycerol or sorbitol and no polysorbates. Therefore, we performed this study to investigate whether sorbitol and glycerol can protect mAb from aggregation under mechanical and interfacial stress such as stirring or shaking to understand if they have the potential to serve as alternatives to polysorbates for stabilizing antibodies. The greater aggregation observed in hydrophobically coated vials under shaking needs to be further investigated.

Methods: A model IgG1 monoclonal antibody was formulated at 1 mg/mL in 20 mM histidine buffer (pH 6.0), with or without glycerol (16mg/mL), sorbitol (16mg/mL) or PS80 (1mg/mL). The shaking stress was applied using a reciprocating shaker at 200 rpm for 72 hours and the stirring stress was generated by magnetic stirring at 300 rpm for 90 minutes. For stirring stress, samples were taken at four time points, i.e.0, 30, 60 and 90 minutes. To assess the protective effect of interfacial interactions, basic glass vials and vials coated to have hydrophobic surfaces with a water contact angle around 100° were used. Aggregation and particle information were analyzed using dynamic light scattering (DLS) and flow imaging microscopy (FIM).

Results: Under stirring stress, the micron-sized particles measured by FIM increased markedly in the buffer group (negative control), indicating substantial aggregation caused by stirring over time. In contrast, the PS80 group exhibited minimal changes, suggesting effective stabilization (Figure 1a). Sorbitol and glycerol groups showed even higher concentration of particles indicating that neither sorbitol nor glycerol matched the protective capacity of PS80. The DLS analysis showed no significant differences among groups except for the glycerol, which exhibited the highest total scattering as shown by normalized intensity (Figure 1b). Stirring stress was conducted using vials that had a hydrophobic coating as well. According to FIM analysis, sorbitol and glycerol reduced the concentration of micron-sized particles compared to the negative control, suggesting some protective effect (Figure 2a). Nevertheless, their performance was still inferior to that of PS80. DLS results showed no signs of reduced aggregation provided by addition of either sorbitol of glycerol (Figure 2b). In contrast to stirring stress, shaking stress produced no increases in micron‑sized particles across all excipient groups in basic glass vials (Figure 3a). Notably, when vials were hydrophobically coated, particles rose dramatically in all mAb formulations, while the blank remained low (Figure 3b). Surprisingly, sorbitol, glycerol, and even PS80 all failed to demonstrate a significant protective effect relative to negative control, indicating hydrophobic surface may overwhelm excipients protection during shaking. Further investigation is needed.

Conclusion: Based on our results, sorbitol and glycerol did not effectively prevent mAb aggregation under shaking stress and showed limited protection under stirring stress when vials were hydrophobically coated. However, their stabilizing effect was consistently inferior to that of PS80. These findings suggest that while polyols may offer partial interfacial protection, they are unlikely to serve as standalone alternatives to polysorbates in antibody formulations.

References: 1. Rosenberg, A.S., Effects of protein aggregates: An immunologic perspective. The AAPS Journal, 2006. 8(3): p. E501-E507.
2. Morales, A.M., et al., End-to-End Approach to Surfactant Selection, Risk Mitigation, and Control Strategies for Protein-Based Therapeutics. The AAPS Journal, 2022. 25(1).
3. Sudrik, C.M., et al., Understanding the Role of Preferential Exclusion of Sugars and Polyols from Native State IgG1 Monoclonal Antibodies and its Effect on Aggregation and Reversible Self-Association. Pharm Res, 2019. 36(8): p. 109.

Figure 1. Protective effect of excipients under stirring stress. Plot showed (a) total concentration of micron particles measured by MFI; (b) normalized intensity measured by DLS.

Figure 2. Protective effect of excipients under stirring stress in hydrophobic-coated vials. Plot showed (a) total concentration of micron particles measured by MFI; (b) normalized intensity measured by DLS.

Figure 3. Total concentration of particles among different study groups under shaking stress in (a) basic glass vials and (b) hydrophobic-coated vials.