Investigation on the Combined Effect of Hydroxypropyl Beta-Cyclodextrin (HPβCD) and Polysorbate in Monoclonal Antibody Formulation

Abstract

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.

Keywords: KLEPTOSE® HPβCD; bevacizumab; excipient; hydroxypropyl beta-cyclodextrin; ipilimumab; monoclonal antibody; polysorbate; protein formulation; protein stability.

Figure 1

Bevacizumab size profiling under light and heat stresses. Aggregation (A), fragmentation (B), and monomer recovery (C) of bevacizumab in different formulations under one cycle of light stress. Aggregation (D), fragmentation (E), and monomer recovery (F) of bevacizumab in different formulations under heat stress for four weeks. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 using one-way ANOVA compared to the commercial formulation.

Figure 2

Bevacizumab size profiling under agitation stress for 2 h. Aggregation (A), and monomer recovery (B) of bevacizumab in different formulations under agitation stress. * p < 0.05 and ** p < 0.01 using one-way ANOVA compared to the commercial formulation.

Figure 3

Ipilimumab size profiling under light and heat stresses. Aggregation (A), fragmentation (B), and monomer recovery (C) of ipilimumab in different formulations under light stress. Aggregation (D), fragmentation (E), and monomer recovery (F) of ipilimumab in different formulations under heat stress for two and four weeks. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 using one-way ANOVA compared to the commercial formulation.

Figure 4

Mutual diffusion coefficient, Dm, against bevacizumab concentration plot (A) and K_D values’ plot (B) in the presence of different KLPETOSE^®HPB concentrations.

Figure 5

Dynamic surface tension of KLEPTOSE^® HPB, polysorbate 20, and polysorbate 80 (A) and dynamic surface tension of bevacizumab, bevacizumab with KLEPTOSE^® HPB, and bevacizumab with polysorbate 20 (B) as measured by the maximum bubble pressure technique.

Figure 6

Particle formation with bevacizumab in different formulations under light (A), heat (B), and agitation stresses (C). * p < 0.05 using one-way ANOVA compared to the commercial formulation buffer.

Figure 7

ITC data at 25 °C for titration of PS 80 into KLEPTOSE^® HPB. The concentration of KLEPTOSE^® HPB in the cell (280 μL) was 2 × 10⁻⁶ M, and the PS 80 concentration in the syringe was 2 × 10⁻³ M. N—reaction stoichiometry, K_d—binding affinity, ΔH—enthalpy, ΔG—binding free energy, T—temperature, ΔS—entropy.

Figure 8

Static surface tension of 0%, 1%, 3%, and 5% of KLEPTOSE^® HPB with PS 80 (A) and PS 20 (B).