Reverting Reversible Aggregates into Monomers and Arresting Rate of Aggregation of Bevacizumab.

Situation

The self-association behavior of monoclonal antibodies (mAbs) is concentration-dependent¹. Increase in aggregate level is often observed during concentration and buffer exchange steps. Reversible aggregates may subsequently aggregate further to the irreversible aggregated state². It is hypothesized that hydroxypropyl-ß-cyclodextrin (HPßCD) can interact with protein to interfere with the self-association process, thereby reducing reversible aggregates.

Challenge

In this work, bevacizumab (Avastin®), a monoclonal IgG1 antibody was chosen as the model protein as it is known to aggregate in both reversible and non-reversible aggregate forms. The study aimed to investigate the effect of two HPßCD (KLEPTOSE® HPB, with MS=0.65 and KLEPTOSE® HP, with MS=0.9) on bevacizumab aggregation during (i) buffer exchange and concentration step and (ii) formulation stability. 

Solution

Bevacizumab (25 mg/mL) formulated in phosphate buffered saline (PBS) at pH 6.2 was purchased from BOC Sciences (NY, USA). The two biopharma grade HPßCD (KLEPTOSE® HP and KLEPTOSE® HPB) were from Roquette Frères (Lestrem, France).  

(I) Buffer exchange and concentration steps (Figure 1): 

• Bevacizumab in PBS was first diluted 1:1 and then buffer-exchanged into 50 mM sodium phosphate buffer, pH 6.2 in the absence or presence of 100 mM KLEPTOSE® HP or HPB using preparative size-exclusion chromatography column (Sephadex G-25 PD-10). 
• An ultrafiltration step using Amicon® ultra centrifugal filters (MWCO= 30KDa) was then performed to concentrate the protein solutions to 50 mg/mL.

(II) Formulation stability (Figure 2)

• The commercial formulation (CF) of bevacizumab (Avastin®) contains 25 mg/mL bevacizumab in 50 mM sodium phosphate buffer at pH 6.2, 60 mg/mL α,α-trehalose dihydrate and 0.4 mg/mL polysorbate 20. 
• In this study, bevacizumab was formulated at 25 mg/mL protein concentration in 50 mM sodium phosphate buffer, pH 6.2 with varying concentration of HPßCD and compared with control formulations: (i) buffer only and (ii) CF. 

Results And Discussions

Buffer exchange and concentration steps

In both buffer exchange and ultrafiltration steps, approximately 25% reduction of aggregate was observed in the presence of KLEPTOSE® HP or HPB (Figure 3).

Reduction in reversible aggregates

At T0, addition of KLEPTOSE® HP or HPB in formulations appeared to reduce reversible aggregates in a concentration-dependent manner (Figure 4). 

Potential interaction between bevacizumab and KLEPTOSE® HP or KLEPTOSE® HPB may interfere with the self-association of bevacizumab, thereby reducing the formation of reversible aggregates.

Formulation stability

Formulations containing KLEPTOSE® HP or HPB exhibited lower rates of aggregation, as compared to commercial formulation during agitation stress (Figure 5A).
When subjected to thermal stress (Figure 5B), formulations containing KLEPTOSE® HP or HPB exhibited lower rates of aggregation, as compared to commercial formulation (containing 60 mg/mL Trehalose).

Conclusion

• KLEPTOSE® HP and HPB were able to reduce the formation of reversible aggregates in bevacizumab formulations. 
• Addition of KLEPTOSE® HP or HPB in buffer was beneficial in reducing aggregates in buffer exchange and concentration steps. 
• KLEPTOSE® HP and HPB were also effective in reducing both agitation and thermal stress-induced aggregation, potentially extending the shelf life of bevacizumab formulation.