Monitoring of Multiple Product Quality Attributes of Biologics using Stable Isotope Labeled Internal Standard and Mass Spectrometry

With the implementation of the quality by design (QbD) concept to drug development, monitoring product quality attributes (PQAs) of biologics during process and product development has become increasingly critical. Mass spectrometry has been widely used for quantification of modifications in therapeutic proteins. In addition the use of internal standards provide unique benefits for accurate and precise quantification, since all variability in sample handling is minimized as the internal standard is introduced into the sample for analysis prior to any sample handling steps, such as proteolytic digestion and chromatographic separation, etc. Here, we describe the introduction by in vivo metabolic labeling of a stable isotope to produce a stable isotope labeled internal standard (SILIS) protein with 15N enriched media. The SILIS protein is the foundation for a novel strategy for automatically monitoring multiple PQAs in a single LC/MS analysis. The 15N labeled biologics was used as an internal standard to monitor multiple PQAs of in-process samples. Precise and accurate quantitation was achieved on a Thermo Scientific LTQ Orbitrap Velos high-resolution mass pectrometer. MassAnalyzer, an in-house developed software at Amgen, was used for automated identification and quantitation of multiple modifications identified in a single peptide map as part of process and product development.