Screening techniques for monitoring the sub-visible particle formation of free fatty acids in biopharmaceuticals.

Free fatty acid (FFA) particles that originate from the enzymatic hydrolysis of polysorbate (PS) via co-purified host cell proteins generally appear abruptly in drug products during real-time (long-term) storage. Efforts were taken to understand the kinetics of FFA particle formation, aiming for a mitigation strategy. However, it is rather challenging particularly in the sub-visible particle (SVP) range, due to either the insufficient sensitivity of the analytical techniques used or the interference of the formulation matrices of proteinaceous drug products. In this study, we examined the feasibility of Raman microscopy, backgrounded membrane imaging (BMI) and total holographic characterization (THC) on the detection of FFA sub-visible particles (SVPs). The results indicate that THC is the most sensitive technique to track their occurrence during the course of PS hydrolysis. Moreover, with this technique we are able to distinguish different stages of FFA particle formation in the medium. In addition, a real time stability study of a biopharmaceutical was analyzed, demonstrating the viability of THC to monitor SVPs in a real sample and correlate it to the visible particles (VPs) occurrence.

Quantifying methionine sulfoxide in therapeutic protein formulation excipients as sensitive oxidation marker.

Methionine is a common excipient used in therapeutic protein liquid formulations as stabilizer and antioxidant. The oxidation of methionine to methionine sulfoxide can be regarded as a sensitive marker of oxidative stress for drug product storage conditions. In this study, a sensitive HPLC method for the quantification of methionine sulfoxide in formulated protein product was developed and qualified according to regulatory requirements using a SIELC® Primesep 100 column with UV detection. The separation involves a mixed-mode mechanism including reversed phase and cationic exchange modalities. The operating range of the method was established between 1 µM and 35 µM of methionine sulfoxide. In this testing range, the method was shown to be linear (R2 > 0.99), accurate (Recovery 92.9 - 103.6%, average recovery = 99.8 ± 1.4%) and precise (intermediate precision at LoQ, CV = 2.9%). The developed test system was successfully applied to study the effects of temperature and storage conditions on methionine sulfoxide formation in complex therapeutic antibody formulations.

Application of affinity capillary electrophoresis for charge heterogeneity profiling of biopharmaceuticals.

Charge heterogeneity profiling is important for the quality control (QC) of biopharmaceuticals. Because of the increasing complexity of these therapeutic entities [1], the development of alternative analytical techniques is needed. In this work, flow-through partial-filling affinity capillary electrophoresis (FTPFACE) has been established as a method for the analysis of a mixture of two similar monoclonal antibodies (mAbs). The addition of a specific ligand results in the complexation of one mAb in the co-formulation, thus changing its migration time in the electric field. This allows the characterization of the charged variants of the non-shifted mAb without interferences. Adsorption of proteins to the inner capillary wall has been circumvented by rinsing with guanidine hydrochloride before each injection. The presented FTPFACE approach requires only very small amounts of ligands and provides complete comparability with a standard CZE of a single mAb.