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Process analytical technology (PAT) is critical for the manufacture of high-quality biologics as it enables continuous, real-time and on-line/at-line monitoring during biomanufacturing processes. The conventional analytical tools currently used have many restrictions to realizing the PAT of current and future biomanufacturing. Here we describe a nanofluidic device for the continuous monitoring of biologics' purity and bioactivity with high sensitivity, resolution and speed. Periodic and angled nanofilter arrays served as the molecular sieve structures to conduct a continuous size-based analysis of biologics. A multiparameter quality monitoring of three separate commercial biologic samples within 50 minutes has been demonstrated, with 20 µl of sample consumption, inclusive of dead volume in the reservoirs. Additionally, a proof-of-concept prototype system, which integrates an on-line sample-preparation system and the nanofluidic device, was demonstrated for at-line monitoring. Thus, the system is ideal for on-site monitoring, and the real-time quality assurance of biologics throughout the biomanufacturing processes.

Polysorbate is widely used to maintain stability of biotherapeutic proteins in pharmaceutical formulation development. Degradation of polysorbate can lead to particle formation in drug products, which is a major quality concern and potential patient risk factor. Enzymatic activity from residual host cell enzymes such as lipases and esterases plays a major role for polysorbate degradation. Their high activity, often at very low concentration, constitutes a major analytical challenge in the biopharmaceutical industry. In this study, we evaluated and optimized the activity-based protein profiling (ABPP) approach to identify active enzymes responsible for polysorbate degradation. Using an optimized chemical probe, we established the first global profile of active serine hydrolases in harvested cell culture fluid (HCCF) for monoclonal antibodies (mAbs) production from two Chinese hamster ovary (CHO) cell lines. A total of eight known lipases were identified by ABPP with enzyme activity information, while only five lipases were identified by a traditional abundance-based proteomics (TABP) approach. Interestingly, phospholipase B-like 2 (PLBL2), a well-known problematic HCP was not found to be active in process-intermediates from two different mAbs. In a proof-of-concept study with downstream samples, phospholipase A2 group VII (PLA2G7) was only identified by ABPP and confirmed to contribute to polysorbate-80 degradation for the first time. The established ABBP approach is approved to be able to identify low-abundance host cell enzymes and fills the gap between lipase abundance and activity, which enables more meaningful polysorbate degradation investigations for biotherapeutic development.

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