Influence of methionine oxidation on the aggregation of recombinant human growth hormone

Oxidation of methionine (Met) residues is one of the major chemical degradations of therapeutic proteins. This chemical degradation can occur at various stages during production and storage of a biotherapeutic drug. During the oxidation process, the side chain of methionine residue undergoes a chemical modification, with the thioether group substituted by a sulfoxide group. In previous papers, we showed that oxidation of the two most accessible methionine residues of recombinant human growth hormone (r-hGH), Met14 and Met125, has no influence on the conformation of the protein [1]. However, the oxidized r-hGH is less thermally stable than the native protein [2]. In the current work, the consequences of the oxidation of these two methionine residues on the aggregation of r-hGH were investigated. The aggregation properties and kinetics of the native and oxidized r-hGH were measured in different buffers with both spectroscopic and chromatographic methods. Stabilities of oxidized and non-oxidized r-hGH were studied after storage at 37 °C and freeze/thawing cycles. Methionine oxidation influenced the aggregation properties of r-hGH. In accelerated stability studies at 37 °C, oxidized hormone aggregated more and faster than non-oxidized hormone. In freezing/thawing stability studies, it was found that oxidized r-hGH was less stable than its non-oxidized counterpart. In case of hGH, we have shown that chemical degradations such as oxidation can affect its physical stability and can induce aggregation.

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