Arresting Rate of Aggregation During Agitation and Thermal Stress Using IgG as a Model Protein.

Situation

Protein instability is a major challenge to overcome during manufacturing, formulation, transportation and storage of biopharmaceuticals¹. The selection of appropriate excipients to stabilize protein drugs during formulation development is critical in ensuring product stability against various physical stresses throughout its recommended shelf life.

Challenge

In this study, we evaluated the role of hydroxylpropyl ß-cyclodextrin in the stabilization of therapeutic proteins. The stabilizing effects of two hydroxypropyl ß-cyclodextrins (KLEPTOSE® HPB, with MS=0.65 and KLEPTOSE® HP, with MS=0.9) against agitation and thermal stresses on human plasma IgG were investigated. A two-prong approach, using nanoDSF (Differential Scanning Fluorimetry) and size-exclusion chromatography (SEC) HPLC was employed to establish validation.

Solution

Lyophilized human plasma IgG was purchased from BOC Sciences (NY, USA) and reconstituted in purified water at 10 mg/mL. A buffer-exchange step was performed using PD-10 desalting columns (GE Healthcare Life Science, Illinois, USA) to exchange out original formulation into a base buffer of 25 mM sodium phosphate buffer, pH 6.8. The two biopharma grade HPßCD (KLEPTOSE® HP and KLEPTOSE® HPB) were from Roquette Frères (Lestrem, France). All other chemicals were purchased from Merck KGaA (Darmstadt, Germany).
Human plasma IgG was formulated at 5 mg/mL protein concentration in two levels of HPßCD (i.e., 20 mM and 100 mM) and compared with control formulations: (i) buffer only, (ii) 0.05 % w/v Tween ® 80 and (iii) 60 mg/mL trehalose. Formulations were subjected to shaking stress at 1400 rpm using an orbital shaker and thermal stress at 40°C in an oven, respectively. Samples were then analyzed at specific time-points using nanoDSF and SEC. 

Results

Surface activity of hydroperoxyde-ß-cyclodextrins

• KLEPTOSE® HP and HPB exhibited surface activity (Figure 1) and can potentially compete with protein at the air-water interface during agitation, inhibiting protein from aggregation. 

Agitation stress

• During agitation stress, lower % unfolding (i.e., lower F350/330 ratio) was observed in formulations containing KLEPTOSE® HP or HPB as compared to controls containing Tween 80 (Figure 2A). 
• When analyzed using SEC, formulations containing KLEPTOSE® HP and HPB were able to inhibit fragmentation, resulting in a significantly higher % soluble monomer recovered as compared to controls (Figure 2B). 

Thermal stress

• During thermal ramp, NanoDSF results showed that KLEPTOSE® HP and HPB (100 mM) reduced the relative amount of protein aggregation and increased aggregation onset temperature (Figure 3A).
• Stability study at 40°C also showed that 100 mM KLEPTOSE® HP and HPB were superior in aggregation reduction as compared to trehalose (Figure 3B). 

Conclusion

• KLEPTOSE® HP and HPB are surface active and can serve as a functional alternative to surfactants in protein formulations.
• Anti-aggregation properties of KLEPTOSE® HP and HPB confirmed using orthogonal methods (nanoDSF and SEC).
• KLEPTOSE® HP and HPB were effective in reducing both agitation and thermal stress-induced aggregation.
• This dual function stabilization property makes hydroxypropyl ß-cyclodextrin a promising excipient to be explored for therapeutic proteins shelf life extension. 

References

1. Steinmeyer, D.E. and E.L. Mccormick, The art of antibody process development. Drug Discovery Today, 2008. 13(13-14): p. 613-18.

Disclaimer

KLEPTOSE® is a registered trademark of Roquette Frères.
Tween® is a registered trademark of Croda Americas LLC.