Biopharmalynx v1

Peptide mapping, which involves enzymatic digestion and tandem MS, was performed to analyze the overall amino acid sequence, post-translational modifications (oxidation and deamidation, etc.), and disulfide bond by LC-ESI-MS.
Eculizumab (SB12 and RP) samples were solubilized with 8 M urea. The denatured samples were reduced by 1 M DTT, and 1 M iodoacetamide (IAA) was then added for alkylation. The samples were 50 mM Tris-HCl buffer (pH 7.8) exchanged for enzyme digestion and digested with the endoprotease trypsin (Roche). For the disulfide linkage analysis, the reducing step was not performed and the non-reduced samples were also digested with trypsin. The digested samples were loaded on a BEH300 C18 reverse-phase column (Waters) connected to Waters UPLC-MS. Data acquisition and processing was performed using MassLynx v4.1 (Waters) and/or BiopharmaLynx v1.2 software (Waters).

Intact mass analysis was performed to determine MW by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS).
Glycosylated and deglycosylated whole protein mass, glycosylated and deglycosylated HC mass, and LC mass of eculizumab (SB12 and RP) were measured using a mass spectrometer after treatment or non-treatment of 1 M 1,4-Dithiothreitol (DTT) and/or PNGase F (NEB). The digested samples were loaded on a BEH300 C4 reverse-phase column (Waters) connected to Waters ultra performance liquid chromatography (UPLC)-MS. Data acquisition and processing was performed using MassLynx v4.1 (Waters) and/or BiopharmaLynx v1.2 software (Waters).

Peptide mapping was performed to analyze structural integrity, including post-translational modifications and disulfide bond by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC–ESI–MS/MS). To achieve denaturation and reduction, ranibizumab was mixed with 8 M urea and 1 M dithiothreitol; 1 M iodoacetamide was added. The sample was digested with trypsin (11047841001, Roche, Basel, Switzerland), Lys-C (90051, Thermo scientific, Waltham, MA, USA), and Asp-N (11058541103, Roche). For the disulfide linkage analysis, the reducing step was not performed, and the non-reduced samples were digested with trypsin. The digestion samples underwent reverse-phase ultra performance liquid chromatography-mass spectrometry (RP-UPLC-MS) using a BEH300 C18 column (186003687, Waters, Milford, MA, USA) at 60 °C. Peptides were eluted by a linear gradient at a flow rate of 0.3 ml/min for 100 min. Data were collected and processed by MassLynx v4.1 (Waters) and/or BiopharmaLynx v1.2 (Waters).