Figure 2. Flow path of the PAK single-use flow kit for individual process steps (chromatography, virus inactivation, membrane, filtration). Includes pH, conductivity (Cond), and pressure (P) instrumentation.
The 500mL flow kits are 3D-printed and include sensors (pH, conductivity, UV280nm, UV300nm, pressure), in-line mixers for buffer addition (pH titration, conductivity adjustment, in-line dilution), a vented mixing vessel and a sample port (Figure 2).

Sterilization and Sanitization Pre-Run

Bioburden control of the continuous purification process required a combination of sanitization and sterilization methods. The Pilot PAK flow kits and associated tubing assemblies were provided gamma-irradiated. Chromatography columns (MabSelect PrismA® used for capture step and SEC column used for VI step) were sanitized with 1N sodium hydroxide during automated start-up sequences, along with the associated flow paths that included pH, conductivity and UV instrumentation. The 0.2µm sterile filter and the Mustang Q membrane were sanitized offline per the vendor specifications with 1N sodium hydroxide prior to use. Glass vessels used as product collection tanks, situated after the perfusion bioreactor and capture step, were autoclaved with tubing. Consumable components such as bags and tubing were gamma irradiated or autoclaved. Sanitary quick connects were used to make any connections that were not sanitized prior to use.

Cell Culture

Several 3L bioreactors were used to generate recombinant IgG via a Chinese Hamster Ovary (CHO) cell line. These individual 3L bioreactors were harvested using coupled Tangential Flow Filtration (TFF), from which they were combined into 50 and 100L bags according to their titers. The titer profile for the 14-day study presented here was generated by ordering the bags based on their titer to represent a typical 50L perfusion bioreactor process. These bags were then welded to the flow path and pumped at a rate of 50L/day into a 10L glass vessel before the dual-column chromatography step. The titer of the material used ranged from 0.2 – 2.34 g/L and totaled 490g of harvested mAb.

Dual Column Chromatography for Protein A Capture

The target molecule was captured with two 4.4 cm diameter x 7.7 cm height chromatography columns using MabSelect PrismA® resin at a capacity of 60 g/L. The columns for the capture step were operated using a dual-column strategy which operated in a typical bind and elute methodology, enabling a continuous load and discontinuous product elution. The size of the columns is optimized for protein mass and process time, resulting in higher resin utilization. Parallel column operation maintains one of the two columns in the load phase at all times. While one column is in the load phase, the other column cycles through the process equilibration, wash, elution, strip and sanitization buffers (Figure 3). The columns were loaded to 60 g/L every cycle; the load duration and frequency of elution varied with the change in titer (load volume was manually input into the PAK automation control screen daily). The elution collection criteria was specified as 5 column volumes (CVs), and collection began after the completion of the first CV. The eluate was collected in a 10L glass vessel downstream of the two columns.