Amino acid sequence analysis and structural characterization of asparaginase and pegylated asparaginase.

A biotech company is currently developing a "biosimilar" asparaginase product. In order to support the development, initial characterization has been performed on commercially available Elspar (asparaginase) and Oncaspar (pegylated asparaginase). The amino acid sequence of El spar and Oncaspar were determined by tryptic peptide map/mass spectrometry. For Oncaspar, pegylation of amine groups throughout the polypeptide chain altered the tryptic digestion pattern and peptide recovery on the map. Mass analysis of unpegylated peptides recovered in the map indicated that the amino acid sequence of Oncaspar was identical to that of Elspar. Asparaginase is a homotetramer linked by non-covalent interactions; the subunit mass resolved on C4 reversed phase (RP) - HPLC was obtained through on-line mass spectrometry (MS) and matched the sequence derived from peptide mapping/MS. The sulfhydryl content of both Elspar and Oncaspar was measured using Ellman's assay; the levels of unpaired cysteines were negligible for both samples. The subunit mass of Elspar indicated the presence of one disulfide bond in each subunit, and non-reduced peptide mapping of both Elspar and Oncaspar confirmed a disulfide bond between Cys77 and Cys 105. The heterogeneity in pegylation levels and loci, as well as, the PEG mass disparity itself, made intact mass measurement and assignment of pegylated peptides impossible. Through size exclusion chromatography coupled with on-line multi-angle laser light scattering (SEC-MALLS), the average molar mass of Oncaspar was estimated to be 280 kDa, indicating that the average degree of pegylation was approximately 28 PEGs per asparaginase homotetramer. An asparaginase sample from the company was subjected to primary structural analysis. Amino acid differences at three positions were observed in comparison to Elspar.