Polysorbates (PS 20 and PS 80) are the most widely used surfactants in biopharmaceutical formulations to protect proteins from denaturation, aggregation, and surface adsorption. To date, around 70% of marketed therapeutic antibodies contain either PS 20 or PS 80 in their formulations. However, polysorbates are chemically diverse mixtures, which are prone to degradation by oxidation and hydrolysis to produce peroxides and fatty acids, which, in turn, induce protein oxidation, aggregation, and insoluble particle formation. These will negatively impact protein quality and stability. Thus, polysorbate degradation has emerged as one of the major challenges in the development and commercialization of therapeutic protein products. KLEPTOSE^® HPβCD (hydroxypropyl beta-cyclodextrin), a new multifunctional excipient, has been shown to provide protein stabilization functions in biopharmaceutical downstream processes and in their final formulations. This study aims to evaluate HPβCD, a new molecule of its class, against polysorbates as a stabilizer in biologics formulations. In this study, the chemical stability of KLEPTOSE^® HPβCDs is compared with polysorbates (20 and 80) under various stress conditions. When subjected to heat stress, HPβCDs show little change in product recovery (90.7–100.7% recovery for different HPβCDs), while polysorbates 20 and 80 show significant degradation, with only 11.5% and 7.3% undegraded product remaining, respectively. When subjected to other chemical stressors, namely, autoclave, light, and oxidative stresses, HPβCD remains almost stable, while polysorbates show more severe degradation, with 95.5% to 98.8% remaining for polysorbate 20 and 85.5% to 97.4% remaining for polysorbate 80. Further, profiling characterization and degradation analysis reveal that chemical structures of HPβCDs remain intact, while polysorbates undergo significant hydrolytic degradation and oxidation. Lastly, the physicochemical stability of monoclonal antibodies in formulations is investigated. When subjected to light stress, adalimumab, as a model mAb, formulated in the presence of HPβCD, shows a significant decrease in protein aggregation, and superior monomer and total protein recovery compared to PS 80-containing formulations. HPβCD also reduces both agitation and thermal stress-induced protein aggregation and prevents subvisible particle formation compared to PS 80.

Monoclonal antibodies require careful formulation due to their inherent stability limitations. Polysorbates are commonly used to stabilize mAbs, but they are prone to degradation, which results in unwanted impurities. KLEPTOSE^® HPβCD (hydroxypropyl beta-cyclodextrin) has functioned as a stable stabilizer for protein formulations in our previous research. The current study investigates the collaborative impact of combining polysorbates and HPβCD as excipients in protein formulations. The introduction of HPβCD in formulations showed it considerably reduced aggregation in two model proteins, bevacizumab and ipilimumab, following exposure to various stress conditions. The diffusion interaction parameter revealed a reduction in protein–protein interactions by HPβCD. In bevacizumab formulations, the subvisible particle counts per 0.4 mL of samples in commercial formulations vs. formulations containing both HPβCD and polysorbates subjected to distinct stressors were as follows: agitation, 87,308 particles vs. 15,350 particles; light, 25,492 particles vs. 6765 particles; and heat, 1775 particles vs. 460 particles. Isothermal titration calorimetry (ITC) measurement indicated a weak interaction between PS 80 and HPβCD, with a K_D value of 74.7 ± 7.5 µM and binding sites of 5 × 10^–3. Surface tension measurements illustrated that HPβCD enhanced the surface activity of polysorbates. The study suggests that combining these excipients can improve mAb stability in formulations, offering an alternative for the biopharmaceutical industry.

Background/Objectives: Low-pH treatment of monoclonal antibodies (mAbs) is commonly employed to inactivate enveloped viruses following Protein A capture chromatography. However, this treatment can sometimes induce conformational changes and aggregation in pH-sensitive proteins. This study investigates the stabilizing effects of four compendial excipients—mannitol, sorbitol, trehalose, and sucrose—on aggregation, as well as on the conformational and colloidal stability of pembrolizumab (IgG₄) and denosumab (IgG₂) in the context of low-pH viral inactivation (VI) operations. Methods: Following Protein A chromatography, pembrolizumab and denosumab were subjected to low-pH incubation and neutralization to mimic the conditions encountered in the VI step. Excipients (10% w/v) were added to the eluate, and aggregation was assessed post-neutralization. Effects on conformational stability (measured by thermal unfolding) and colloidal stability (via diffusion interaction parameter K_D) were investigated. Additionally, the impact of excipients on the dynamic binding capacity of the cation-exchange (CIEX) column was evaluated. Results: All tested excipients significantly reduced aggregation of both mAbs during low-pH incubation. For instance, mannitol reduced aggregation by sixfold in pembrolizumab and by threefold in denosumab. These additives enhanced conformational stability, as evidenced by the increased melting temperatures (Tm), with a pronounced stabilizing effect on the CH2 domain at low pH. Furthermore, the presence of sugars and polyols was associated with higher K_D values, indicating improved colloidal stability under acidic conditions. Importantly, adding these sugars or polyols did not impact the dynamic binding capacity of the CIEX column used in subsequent processing. Conclusions: This study offers valuable insights and supports the rational use of polyols and sugars for stabilizing mAbs during the low-pH VI step.