Chemical Stability of Insulin Formulations

PHARMACEUTICAL CONCERNS

Chemical Stability of Insulin Formulations

Insulin has two primary routes of chemical degradation upon storage and use: hydrolytic transformation of amide to acid groups and formation of covalent dimers and higher order polymers. Primarily the pH, the storage temperature, and the components of the specific formulation influence the rate of formation of these degradation products. The purity of insulin formulations is typically assessed by high-perfor-mance liquid chromatography using reversed-phase and size exclusion separation modes (USP Monographs: Insulin, 2006). In acidic solution, the main degradation reaction is the transformation of asparagine (Asn) at the terminal 21 position of the A-chain to aspartic acid. This reaction is relatively facile at low pH, but is extremely slow at neutral pH (Brange et al., 1992b). This was the primary degrada-tion route in early soluble (acidic) insulin formula-tions. However, the development of neutral solutions and suspensions has diminished the importance of this degradation route. Stability studies of neutral solutions indicate that the amount of A21 desamido insulin does not change upon storage. Thus, the relatively small amounts of this bioactive material present in the formulation arise either from the source insulin or from pharmaceutical process operations.

The deamidation of the Asn^B3 of the B-chain is the primary degradation mechanism at neutral pH. The reaction proceeds through the formation of a cyclic imide that results in two products, aspartic acid (Asp) and iso-aspartic acid (iso-Asp) (Brennan and Clarke, 1994). This reaction occurs relatively slowly in neutralsolution (approximately 1/12 the rate of A21 desamido formation in acid solution) (Brange et al., 1992b). The relative amounts of these products are influenced by the flexibility of the B-chain, with approximate ratios of Asp:iso-Asp of 1:2 and 2:1 for solution and crystalline formulations, respectively. As noted earlier, the use of phenolic preservatives provides a stabilizing effect on the insulin hexamer that reduces the formation of the cyclic imide, as evidenced by reduced deamidation. The rate of formation also depends on temperature; typical rates of formation are approximately 2% per year at 5LC. Studies have shown B3 deamidated insulin to be essentially fully potent (Chance, 1995).

High-molecular-weight protein (HMWP) pro-ducts form at both storage and room temperatures. Covalent dimers that form between two insulin molecules are the primary condensation products in marketed insulin products. There is evidence that insulin-protamine heterodimers also form in NPH suspensions (Brange et al., 1992a). At higher tempera-tures, the probability of forming higher order insulin oligomers increases. The rate of formation of HMWP is less than that of hydrolytic reactions; typical rates are less than 0.5% per year for soluble neutral Regular insulin formulations at 5LC. The rate of formation can be affected by the strength of the insulin formulation or by the addition of glycerol as an isotonicity agent. The latter increases the rate of HMWP formation presumably by introducing impurities such as glycer-aldehyde. HMWP formation is believed to also occur as a result of a reaction between the N-terminal B1 phenylalanine amino group and the C-terminal A21 asparagine of a second insulin molecule (Darrington and Anderson, 1995).

Disulfide exchange leading to polymer formation is also possible at basic pH; however, the rate for these reactions is very slow under neutral pH formulation conditions. The quality of excipients such as glycerol is also critical because small amounts of aldehyde and other glycerol-related chemical impurities can accelerate the formation of HMWP. The biopotency of HMWP is significantly less (1/10 to 1/5 of insulin) than monomeric species (Chance, 1995).

Unfortunately, no chemical stability data has been published with regard to the insulin analog formulations containing insulin lispro, insulin aspart, insulin glulisine, insulin glargine, or insulin detemir; however, it is reasonable to presume that similar chemical degradation pathways are present to varying extents in these compounds. Moreover, since some analogs are formulated under acidic conditions, e.g., Lantus is formulated at pH 4.0, or have been modified with hydrophobic moieties, e.g., Levemir, it is reason-able to presume that alternate chemical degradation pathways may be operable.