(W1230-07-37) Hydroxypropyl Beta-Cyclodextrin as a Multifunctional Excipient for Downstream Processing and Formulation of Monoclonal Antibodies

Purpose: Monoclonal antibodies (mAbs) have revolutionized modern medicines in the fast-growing biopharmaceuticals sector. However, the inherent instability of protein molecules presents significant challenges in downstream processing, final formulation, and drug delivery. For example, particle formation or protein aggregation can occur during unit operations such as ultrafiltration/diafiltration (UF/DF). Also, the inherent stability limitations of mAbs will require careful formulation. In this work, we investigated hydroxypropyl beta-cyclodextrin (HPβCD) as a multifunctional excipient to reduce particle formation during UF/DF of different mAbs and in subsequent formulation. The stabilizing effect of KLEPTOSE® HPβCD was compared with polysorbates and their combinations as excipients in mAbs formulations were evaluated.

Methods: Bevacizumab was purchased from BOC Sciences (NY, USA). Adalimumab was produced in Chinese hamster ovary (CHO) K1 cells and purified in-house. Biopharma grade mannitol (PEARLITOL®) and three different HPβCD products, namely, KLEPTOSE® HP (biopharma grade), KLEPTOSE® HPB (biopharma grade), and KLEPTOSE® HPB-LB (parenteral grade), were from Roquette Frères (Lestrem, France). Trehalose dihydrate and sucrose were from Acros Organics and Calbiochem, respectively. UF/DF study: Ultrafiltration/diafiltration recirculation experiments of adalimumab were conducted using LabscaleTM TFF System with Pellicon® XL50 cassette with Biomax® 30 kDa membrane. To study the effect of KLEPTOSE® HPB, 2 mg/mL adalimumab in citrate buffer, pH 5.2, with and without 25 mM KLEPTOSE® HPB was recirculated in the TFF system for 4 hours. Retentates were analyzed at every hour with turbidity meter, Dynamic Light Scattering (DLS)and Size exclusion Chromatography (SEC-HPLC). Force degradation studies of HPβCD products and polysorbates: Chemical stability of KLEPTOSE® HP, HPB, and HPB-LB, and polysorbates (PS) in aqueous medium was evaluated under various stressed conditions such as thermal, light, autoclave and oxidation stresses. Quantification and characterization of the parent chemicals and their degradants were evaluated using UPLC system with a QDa detector and Dionex HPLC–CAD system. Antibody Formulation and Stability Studies: The efficiency of HPβCD and PS 20 in preventing protein aggregation and particle formation was studied under various stress conditions. All the formulations were subjected to agitation stress (i.e. stirred at 200 rpm for 2 h), heat stress (i.e. 40 °C /75% RH for 2–4 weeks) and light stress (i.e. exposed to white light for 1.2 million Lux hours and UV light for 200 watt-hours per square meter 4). All samples were prepared in duplicates.

Results: After 4 hours of UF/DF recirculation, an increase in turbidity of the adalimumab solution was observed over time. The addition of 25 mM KLEPTOSE®HPB significantly reduced the rate of turbidity increase in adalimumab solution and the final turbidity was more than 4 times lower compared to the control sample (Figure 1A). Additionally, we observed a lower relative intensity of larger particles in the presence of KLEPTOSE® HPB (Figure 1B). This shows that KLEPTOSE® HPB was effective in reducing UF/DF-induced particle formation in adalimumab solution. In the bevacizumab formulation study (Figure 2A), KLEPTOSE® HPB-containing (5% w/v and 10% w/v) formulations presented significantly lower aggregation levels compared to the commercial formulation under light stress (6.92% and 6.82% vs. 11.28%) and heat stress (10.59% and 9.39% vs. 18.88%). The formulations containing both KLEPTOSE® HPB and polysorbate also displayed significantly lower aggregation levels compared to the commercial formulation. The protective effect of the combination was further evidenced in subvisible particle counts. Formulations containing both KLEPTOSE® HPB and polysorbates consistently displayed lowest subvisible particle numbers in all subjected stresses compared to commercial formulation or KLEPTOSE® HPB alone formulations. Our study also compared the stability of different grades of KLEPTOSE® HPβCD and polysorbates under different stress conditions. When subjected to thermal stress at 40 °C for 24 weeks, different grades of KLEPTOSE® HPβCD showed slight changes in product recovery (100.7% -90.7%), while PS 20 and PS 80 suffered significant degradation with the product recovery of 11.5% and 7.3%, respectively (Figure 3A). When the samples were subjected to autoclave for 5 cycles at 121 °C, oxidation with 0.3% H2O2, and light exposure, both PS 20 and PS 80 suffered around 10% product loss. In contrast to polysorbates, all KLEPTOSE® HPβCDs remained stable when subjected to the same stresses (Figure 3B).

Conclusion: This study has demonstrated the utility of KLEPTOSE® HPβCD as a versatile excipient for UF/DF process and in subsequent protein formulation. KLEPTOSE® HPβCD has been shown to improve protein recovery and reduce particle formation in adalimumab during UF/DF. KLEPTOSE® HPβCD has also been shown to be useful in reducing protein aggregation under heat and light stresses during formulation stability study. Notably, the combination of KLEPTOSE® HPβCD and polysorbates not only significantly reduced antibody aggregation but also reduced the counts of subvisible particles when subjected to multiple stressors, as compared to commercial formulations. Our data presented here also strongly suggests that HPβCD exhibits excellent stability during various stress conditions. These findings have proven KLEPTOSE® HPβCD is a promising excipient to be explored for biotherapeutic downstream processing and formulation applications.

References: 1. Castañeda Ruiz, A. J.; Shetab Boushehri, M. A.; Phan, T.; Carle, S.; Garidel, P.; Buske, J.; Lamprecht, A., Alternative Excipients for Protein Stabilization in Protein Therapeutics: Overcoming the Limitations of Polysorbates. Pharmaceutics 2022, 14 (12), 2575.
2. Dwivedi, M.; Blech, M.; Presser, I.; Garidel, P., Polysorbate degradation in biotherapeutic formulations: identification and discussion of current root causes. International journal of pharmaceutics 2018, 552 (1-2), 422-436.
3. ICH, D. R. G. In Stability testing of new drug substances and products Q1A (R2), Proceedings of the International Conference on Harmonization. Geneva, Switzerland, 2003.
4. Guideline, I. H. T., Stability testing: photostability testing of new drug substances and products. Q1B, Current Step 1996, 4.


Figure 1. Particle formation in 2 mg /mL adalimumab solution after UF/DF recirculation. (A) Turbidity and (B) DLS relative intensity >0.1µm of adalimumab with and without KLEPTOSE® HPB (n=3, mean ±SE).


Figure 2. A. Bevacizumab size profiling under light and heat stresses. B. Particle formation with bevacizumab in different formulations under light, heat, and agitation stresses. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 using one-way ANOVA compared to the commercial formulation.