Simultaneous Method Development and Validation for Quantitative Analysis of Ocrelizumab and Hyaluronidase in Bulk and Pharmaceutical Dosage Forms Using UPLC

Abstract— A simple, rapid, precise, sensitive, and reproducible Ultra PerformanceLiquid Chromatography (UPLC) method has been developed and validated for thequantitative analysis of Ocrelizumab and Hyaluronidase in bulk and pharmaceuticaldosage forms. Chromatographic separation was achieved on a Waters AcquityUPLC system using a Kinetex C18 column (50 mm x 2.1 mm, 1.7 µm) with a mobilephase consisting of Acetonitrile and 0.1% Triethylamine (TEA) buffer adjusted to pH2.5 with Orthophosphoric acid (OPA) in an 80:20 (v/v) ratio. The flow rate was setat 0.5 mL/min, and detection was performed by UV absorption at 260 nm using aphotodiode array (PDA) detector at ambient temperature. The theoretical platecount and tailing factor for Ocrelizumab and Hyaluronidase met standardregulatory criteria (NLT 2000 and <= 2.0, respectively). Relative standard deviation(%RSD) for peak area of all measurements was consistently below 2.0%. Theproposed method was validated according to ICH guidelines and demonstratedsimplicity, economy, suitability, precision, accuracy, robustness, and stabilityindicating properties.