BACKGROUND: Peptide-Fc fusion proteins are inherently heterogeneous and complex molecules. Protein post-translational modifications (PTMs) or truncation can arise during manufacturing or product storage. Some of these product attributes could potentially impact the efficacy or safety of the bio-molecule and are thus classified as critical quality attributes (CQAs). These CQAs should be controlled in order to ensure manufacturing and quality consistency.
METHODS: A subunit UPLC-ToF MS based MAM method was developed for identity test and quantitatively monitored two critical quality attributes (CQAs) resulting from two truncations of that fusion protein (fragment 1 and 2). Three independent laboratories are involved in the method validation according to ICH Q2(R1), ICH Q6B, FDA and NMPA guidance.
RESULTS: This developed method fully meets the pre-defined analytical target profile (ATP), including specificity, accuracy, precision, quantitation limit, linearity, range and robustness. Three independent labs co-validate a UPLC-ToF MS based MAM method for protein drug QC release and stability testing.
CONCLUSIONS: The experimental design of method validation can be a reference for LC-HRMS-based subunit MAM methods that have been widely used in the characterization of antibodies, ADCs and other protein-based biologics. This work paves the way for implementing MAM in QC with more targeted control of product quality.

Succinimide (Asu) is the intermediate for asparagine deamidation in therapeutic proteins, and it can be readily hydrolyzed to form aspartate and iso-aspartate residues. Moreover, Asu plays an important role in the protein degradation pathways, asparagine deamidation, and aspartic acid isomerization. Here, Asu modification with a high abundance in the framework region (FR) of golimumab was first reported, the effect of denaturing buffer pH on the Asu modification homeostasis was studied, and the results revealed that it was relatively stable over a pH range of 6.0-7.0 whereas a rapid decrease at pH 8.0. Then, the peptide-based multi-attribute method (MAM) analyses showed that the Asu formation was at Asn 43 in the FR of the heavy chain. Meanwhile, the efficacy [affinity, binding and bioactivity, complement-dependent cytotoxicity (CDC) activity, and antibody-dependent cell-mediated cytotoxicity (ADCC) activity] and stability of the Asu modification of golimumab were evaluated, and the current results demonstrated comparable efficacy and stability between the Asu low- and high-abundance groups. Our findings provide valuable insights into Asu modification and its effect on efficacy and stability, and this study also demonstrates that there is a need to develop a broad-spectrum, rapid, and accurate platform to identify and characterize new peaks in the development of therapeutic proteins, particularly for antibody drugs.