Structural basis for cyclodextrins' suppression of human growth hormone aggregation

Abstract

Many therapeutic proteins require storage at room temperature for extended periods of time. This can lead to aggregation and loss of function. Cyclodextrins (CDs) have been shown to function as aggregation suppressors for a wide range of proteins. Their potency is often ascribed to their affinity for aromatic amino acids, whose surface exposure would otherwise lead to protein association. However, no detailed structural studies are available. Here we investigate the interactions between human growth hormone (hGH) and different CDs at low pH. Although hGH aggregates readily at pH 2.5 in 1 M NaCl to form amorphous aggregates, the presence of 25 to 50 mM of various ␤-CD derivatives is sufficient to completely avoid this. ␣-and ␥-CD are considerably less effective. Stopped-flow data on the aggregation reaction in the presence of ␤-CD are analyzed according to a minimalist association model to yield an apparent hGH-␤-CD dissociation constant of ∼6 mM. This value is very similar to that obtained by simple fluorescence-based titration of hGH with ␤-CD. Nuclear magnetic resonance studies indicate that ␤-CD leads to a more unfolded conformation of hGH at low pH and predominantly binds to the aromatic side-chains. This indicates that aromatic amino acids are important components of regions of residual structure that may form nuclei for aggregation.

The Effect of Cyclodextrins on Chemical and Physical Stability of Glucagon and Characterization of Glucagon/γ-CD Inclusion Complexes

“…Thus, the ability of CDs to improve the physical stability of glucagon is a consequence of inclusion complex formation, which has also been described with other peptides. 18,24,25,31 In addition, the higher viscosity of the solutions containing CDs may also have affected the physical stability of glucagon by delaying the aggregation in the present study.…”

“…Aromatic side chains of peptides have earlier been demonstrated to be inserted into the cavity of dimethyl-b-CD 7 and natural b-CD. 6,17,18 The side chains of leucines have been shown to be incorporated into the cavity of natural a-and b-CDs 19,20 and dimethyl-a-and dimethyl-b-CDs. 21 Indeed most of the hydrophobic amino acids would fit spatially into the cavity of b-CDs, whereas the steric fit of such amino acids into the cavity of g-CD does not seem to be optimal.…”

“…24 In agreement, there are studies describing the poor effect of g-CD on solubility or stability properties of peptides/proteins. For example, Otzen et al 18 showed that g-CD was much less effective than b-CD in solubilizing human growth hormone. In addition, Banga and Mitra 25 reported that hydroxypropyl-b-CD, b-CD and a-CD prevented aggregation of insulin whereas g-CD did not have any such effect.…”

“…18 CD complexation can isolate the hydrophobic moieties that would otherwise come into intermolecular contact across the interface of two peptide/protein molecules. 31 By preventing such hydrophobic interactions, CDs are able to inhibit the initiation of aggregation.…”

Development of a Transnasal Delivery System for Recombinant Human Growth Hormone (rhGH): Effects of the Concentration and Molecular Weight of Poly-L-arginine on the Nasal Absorption of rhGH in Rats

“…It might be related to the change in three-dimensional structure of rhGH by isoelectric point and low pH. 21,22) Insoluble aggregate was formed when rhGH was mixed with succinate buffer at pH 4.4, but it was not formed in other solution at pH 4.4 (data not shown). Therefore, it seems that the formation is related to other reasons such as sort of solvent, ionic strength.…”