Purpose: Protein aggregation is the major challenge encountered during manufacturing, storage and transportation of biopharmaceuticals. Various pathways to aggregation and one of the most problematic is hydrophobic protein-protein interaction.
Two bio-grade hydroxypropyl-ß-cyclodextrins with different degrees of substitution (KLEPTOSE® HPB BioPharma hydroxypropyl-ß-cyclodextrin, with MS=0.65) and (KLEPTOSE® HP BioPharma hydroxypropyl-ß-cyclodextrin, with MS=0.9) appear to prevent protein aggregation.
Human growth hormone (hGH) aggregates in ionic formulations such as saline (150 mM NaCl) and therefore was used as a protein aggregation model.
A novel approach using high-throughput formulation screening (iFormulate™) and nanoDSF (Differential Scanning Fluorimetry) was used to investigate the role and mechanism of the two bio-grade hydroxypropyl-ß-cyclodextrins on preventing hGH aggregation during thermal stress.
Methods: hGH, a polypeptide of recombinant DNA synthesized in E.coli, was purchased from BOC Sciences as a lyophilized powder and reconstituted at 20 mg solid/ml in phosphate buffered saline (PBS). The protein was diluted to 4 mg/ml into the target formulations of iFormulate™. The formulation design space was assessed using iFormulate™ that utilizes a quadratic response-surface design evaluating four key formulation variables: pH (4-7.6), ionic strength (0-200 mM NaCl), stabilizer (bio-grade hydroxypropyl-ß-cyclodextrins, (0-200 mM) and buffer concentration (10-50 mM). (www.iformulate.net).
Aggregation conditions were defined by exposing hGH in iFormulate™ system to thermal stress at a heating rate of 1.5 C/min from 20 oC to 90 oC. The effect of HP and HPB at concentrations from 0 - 200 mM were evaluated by nanoDSF.
Simultaneous evaluation of Tm (melting temperature of protein unfolding), onset temperature of aggregation, and relative degree of aggregation in various molarity of KLEPTOSE® HPB BioPharma and KLEPTOSE® HP BioPharma (0 mM, 0.125 mM, 0.25 mM, 1.25 mM, 2.5 mM, 5 mM, 10 mM, 25 mM, 50 mM, 100 mM and 200 mM) was performed using differential scanning fluorimetry with nanoDSF instrument (Prometheus NT.Plex) accurately, reproducibly, and precisely.
Prior to thermal stress protein integrity was evaluated by Proteinchip Bioanalyzer.
Results: 1. HP and HPB shows dramatic aggregation inhibition of hGH at concentrations above 100 mM.
2. The DSF results show that in the presence of 100 mM HP or HPB, the onset of aggregation (Figure 1, bottom panel) is significantly delayed to after hGH reaches Tm (50 % denatured) and completely denatured (100 % denatured) around 75.7 oC (Figure 1, top panel).
3. The relative amount of hGH aggregation is also dramatically affected in the presence of 100 mM and eradicated at 200 mM of HP or HPB.
4. The mechanism by which aggregation is inhibited appears to be by protein-HP and protein-HPB interaction. This is shown by the changes in the initial F350/330 ratio in the DSF profile that is indicative of changes in the tertiary conformation of the protein.
Conclusion: 1. Both HP and HPB are effective excipients in preventing hGH aggregation at concentrations above 100 mM.
2. Both the extent of relative aggregation and onset temperature of aggregation of hGH are significantly affected by presence of HP and HPB in the formulation.
3. The data clearly suggests that HP and HPB interact with hGH and may stabilize the protein domains that may cause aggregation.
4. High throughput formulation using iFormulate™ and nanoDSF with the Prometheus NT.Plex instrument provide an effective approach to formulation development and evaluation of novel excipients such as HP and HPB in protein formulations.