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

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.

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Materials

The biopharma-grade mannitol and HPßCD (KLEPTOSE^® HPB) used in this study were from Roquette Frères (Lestrem, France). KLEPTOSE^® HPB has a molar substitution (M.S) of 0.62 and a molecular weight of 1387. α, α-trehalose dihydrate was purchased from Acros Organics (Fair Lawn, NJ, USA). Polysorbate 20 (PS 20) and polysorbate 80 (PS 80), sodium phosphate (monobasic, monohydrate) and sodium phosphate (dibasic, anhydrous), and diethylenetriamine pentaacetate (DTPA) were purchased from Merck KGaA (Darmstadt, Germany). All the chemicals and surfactants were multi-compendia or USP grade. Acetonitrile and methanol (HPLC grade) were purchased from J.T Baker (Phillipsburg, NJ, USA). The monoclonal antibody bevacizumab was purchased from BOC Sciences (Shirley, NY, USA). It was formulated in phosphate buffered saline (PBS) at pH 6.2 at a protein concentration of 25.1 mg/mL. Ipilimumab was produced in Chinese hamster ovary (CHO) cells and purified in-house.

Huang, J.; Hong, S.; Goh, L.Y.H.; Zhang, H.; Peng, T.; Chow, K.T.; Gokhale, R.; Tuliani, V. Investigation on the Combined Effect of Hydroxypropyl Beta-Cyclodextrin (HPβCD) and Polysorbate in Monoclonal Antibody Formulation. Pharmaceuticals 2024, 17, 528. https://doi.org/10.3390/ph17040528

Read also our introduction article on Mannitol here:

excipients formulation