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 [1]. For example, particle formation or protein aggregation can occur during unit operations such as ultrafiltration/diafiltration (UF/DF) [2]. Also, the inherent stability limitations of mAbs will require careful formulation.

Objectives

In this work, we investigated KLEPTOSE® HP, HPB and HPB-LB 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

1. UF/DF study

2. Force degradation studies of HPβCD (KLEPTOSE® HP, HPB, and HPB-LB) and polysorbates

3. Antibody Formulation and Stability Studies

Results

Ultrafiltration/Diafiltration (UF/DF)

Ultrafiltration/diafiltration (UF/DF) processes are known to cause significant stresses to proteins, potentially leading to protein aggregation, particle formation and product losses. Figures 1 and 2 illustrate the efficacy of KLEPTOSE® HPB in mitigating UF/DF-induced particle formation in Human IgG plasma and adalimumab solutions:

• The addition of KLEPTOSE® HPB significantly reduced the rate of turbidity increase in both Human IgG plasma and adalimumab solutions.
• A lower relative intensity of larger particles in the presence of KLEPTOSE® HPB.

Furthermore, KLEPTOSE® HPB also greatly improved protein recovery of human plasma IgG solution as shown in Figure 3.

Figure 1. Turbidity of  (A) 1 mg/mL human plasma IgG solution and (B) 2 mg/ml adalimumab with and without KLEPTOSE® HPB after UF/DF recirculation (n=3, mean ±SE). * 4F denotes final product recovery after flushing from membrane.

Figure 2. Turbidity and % DLS relative intensity of particles >1 μm after ultrafiltration of human plasma IgG solution from 10 to 90 mg/mL, (n=3, mean ±SE). Concentration factor = protein concentration/initial protein concentration.

Figure 3. Protein recovery and aggregate levels in 1 mg /mL human plasma IgG solution after diafiltration. *4F denotes final product recovery after flushing from membrane.

Forced degradation of HPβCD and polysorbates

The chemical stability of KLEPTOSE® HPβCD is compared to that of polysorbates (PS) under various stress conditions. Figure 4 illustrates the stability of HPβCD and polysorbates (PS) under oxidative, light and heat stresses. The results indicate that:

• When subjected to heat stress, PS20 and PS80 undergo tremendous degradation, while KLEPTOSE® HPβCD maintained its stability.
• KLEPTOSE® HPβCD shows superior stability under light, and oxidation stresses than PS20 and PS80.
  
   

Figure 4. Stability study of polysorbates and KLEPTOSE® HPβCD under oxidative, light and heat stresses.

Antibody Formulation and Stability Studies

Figures 5 and 6 illustrate the levels of aggregates obtained under light and heat stresses for bevacizumab and Ipilimumab. The data indicate that KLEPTOSE® HPβCD, either alone, or combination with polysorbates, enhanced the stability of various mAbs by reducing protein aggregation under these various conditions.

Figure 5. Bevacizumab aggregation levels under light and heat stresses. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 using one-way ANOVA compared to the commercial formulation.

Figure 6. Ipilimumab aggregation levels under light and heat stresses. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 using one-way ANOVA compared to the commercial formulation.

Figure 7 presents the particle formation under agitation stress for bevacizumab formulations. It is clearly demonstrated here that KLEPTOSE® HPβCD alone, or combination with polysorbates, helps reduce particle formation caused by agitation stress.

Figure 7. Particle formation with bevacizumab in different formulations under agitation stresses. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 using one-way ANOVA compared to the commercial formulation.

The efficacy of KLEPTOSE® HPβCD with and without polysorbate in reducing mAbs aggregation and particle formation is summarized in tables 1 and 2.

Table 1. Effectiveness of different formulations on reducing aggregation of three mAbs under stresses. Numbers of + represent the degree of effectiveness compared to samples formulated in buffer only.

Table 2. Effectiveness of different formulation on reducing particle formation of three mAbs under stresses. Numbers of + represent the degree of effectiveness compared to samples formulated in buffer only.

Conclusion

This study has demonstrated the utility of KLEPTOSE® HPβCD as a versatile excipient for UF/DF process and in subsequent protein formulation:

• Our findings reveal that HPβCD exhibits excellent chemical stability under various stress conditions, highlighting its potential as a promising excipient for biotherapeutic downstream processing and formulation.
• KLEPTOSE® HPβCD enhances protein recovery and minimizes particle formation during ultrafiltration and diafiltration.
• It effectively reduces protein aggregation under heat and light stress in several monoclonal antibody formulations, as shown in stability studies.
• Notably, when combined with polysorbates, KLEPTOSE® HPβCD significantly decreases antibody aggregation and subvisible particle counts under multiple stressors, outperforming commercial formulations.