Combining process analytical technology (PAT) with continuous production provides a powerful tool to observe and control monoclonal antibody (mAb) fermentation and purification processes. This work demonstrates on-line liquid chromatography (on-line LC) as a PAT tool for monitoring a continuous biologics process and forced degradation studies. Specifically, this work focused on ion exchange chromatography (IEX), which is a critical separation technique to detect charge variants. Product-related impurities, including charge variants, that impact function are classified as critical quality attributes (CQAs). First, we confirmed no significant differences were observed in the charge heterogeneity profile of a mAb through both at-line and on-line sampling and that the on-line method has the ability to rapidly detect changes in protein quality over time. The robustness and versatility of the PAT methods were tested by sampling from two purification locations in a continuous mAb process. The PAT IEX methods used with on-line LC were a weak cation exchange (WCX) separation and a newly developed shorter strong cation exchange (SCX) assay. Both methods provided similar results with the distribution of percent acidic, main, and basic species remaining unchanged over a 2 week period. Second, a forced degradation study showed an increase in acidic species and a decrease in basic species when sampled on-line over 7 days. These applications further strengthen the use of on-line LC to monitor CQAs of a mAb continuously with various PAT IEX analytical methods. Implementation of on-line IEX will enable faster decision making during process development and could potentially be applied to control in biomanufacturing.

For many protein therapeutics including monoclonal antibodies, aggregate removal process can be complex and challenging. We evaluated two different process analytical technology (PAT) applications that couple a purification unit performing preparative hydrophobic interaction chromatography (HIC) to a multi-angle light scattering (MALS) system. Using first principle measurements, the MALS detector calculates weight-average molar mass, Mw and can control aggregate levels in purification. The first application uses an in-line MALS to send start/stop fractionation trigger signals directly to the purification unit when preset Mw criteria are met or unmet. This occurs in real-time and eliminates the need for analysis after purification. The second application uses on-line ultra-high performance size-exclusion liquid chromatography to sample from the purification stream, separating the mAb species and confirming their Mw using a µMALS detector. The percent dimer (1.5%) determined by the on-line method is in agreement with the data from the in-line application (Mw increase of approximately 2750 Da). The novel HIC-MALS systems demonstrated here can be used as a powerful tool for real-time aggregate monitoring and control during biologics purification enabling future real time release of biotherapeutics.

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Hydrogen exchange mass spectrometry (HXMS) coupled to proteolytic digestion has been used to probe the conformation of bovine β-lactoglobulin (BLG), bovine α-lactalbumin (BLA), and human serum albumin (HSA) in solution and while adsorbed to the hydrophobic interaction chromatography media Phenyl Sepharose 6FF. All three proteins show evidence of EX1 exchange kinetics, indicating a loss of stability on the surface. HX protection patterns for all three proteins also indicate that the unfolded form is only partially solvent exposed. The hydrogen-deuterium exchange patterns of BLG and BLA on the surface suggest a structure that resembles each protein's respective solution phase molten globule state. The low stability of Domain II of HSA observed on Phenyl Sepharose 6FF also suggests a link to solution stability because Domain II is frequently cited as the least stable domain in solution unfolding pathways. COREX, an algorithm used to compute protein folding stabilities, correctly predicts solution hydrogen-deuterium exchange patterns for BLG and offers insight into its adsorbed phase stabilities but is unreliable for BLA predictions. The results of this work demonstrate a link between solution-phase local stability patterns and the nature of partially unfolded states that proteins can adopt on HIC surfaces.

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Protein concentration determination is a necessary in-process control for the downstream operations within biomanufacturing. As production transitions from batch mode to an integrated continuous bioprocess paradigm, there is a growing need to move protein concentration quantitation from off-line to in-line analysis. One solution to fulfill this process analytical technology need is an in-line index of refraction (IoR) sensor to measure protein concentration in real time. Here the performance of an IoR sensor is evaluated through a series of experiments to assess linear response, buffer matrix effects, dynamic range, sensor-to-sensor variability, and the limits of detection and quantitation. The performance of the sensor was also tested in two bioprocessing scenarios, ultrafiltration and capture chromatography. The implementation of this in-line IoR sensor for real-time protein concentration analysis and monitoring has the potential to improve continuous bioprocess manufacturing.

Hydrophobic interaction chromatography (HIC) purifies proteins based on their apparent hydrophobicities, making it a valuable tool in downstream purification. However, interactions between buried hydrophobic residues and HIC surfaces can cause conformational changes that lead to compromised chromatographic behavior and yield losses. Recent observations with proteins on ion-exchange chromatography (IEC) surfaces demonstrate that electrostatic interactions may also drive conformational changes.Unfolding of proteins on HIC and IEC surfaces has particular implications for multi-mode ii The utility of HXMS was also demonstrated in identifying the regions involved in the self-association of a therapeutic protein (Protein X). Protein X displays split peak behavior during IEC whereas an analogous protein similar to Protein X does not. HXMS was used to show that a modified region not present on the analogous protein is involved in the intermolecular contact between associated molecules. However, a conformational change was also detected in a binding pocket of the unmodified region suggesting self-association may involve insertion of the modified region from one molecule into this binding pocket of another. iii

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