2.1. End-to-end continuous processing
The schematic and process diagram of the end-to-end continuous process from cell culture to purification that was used in this study is shown in Figure 1. In the process diagram, washing, equilibration, regeneration and replacement of columns and filters before and after each step are shown in gray. Starting from day 12 of cell culture, mAb expressed by the perfusion cell culture was harvested and purified by successively performing Protein A column capture, pooled low pH virus inactivation, and flow-through two column chromatography and virus filtration (total flow-through integrated polishing). In the end-to-end continuous process used in this study, the process stream was only pooled for the low pH virus inactivation process, and this production unit is used to demarcate the batch. In this study, a total of three batches were processed in succession. Impurities and mAb concentration at each step were evaluated off-line (Figure 2).
For the end-to-end continuous process trials, mAb (pI 8.5) expressed by a CHO-MK cell line in perfusion cell culture developed by the Manufacturing Technology Association of Biologics (MAB) at its GMP facility was used. The proprietary process consists of harvest of the perfusion cell culture process by XCell ATF-2 (Repligen) at 1.1 vvd (vessel volume exchanges per day), followed by a continuous capture process on two 1-mL Protein A columns (KANEKA KanCapA 3G, Kaneka Corporation) in periodic counter current (PCC) mode on a Contichrom HPLC 30 (YMC Co., Ltd.) with continuous elution of purified mAb with 60 mM acetate (pH 3.6) buffer in cycles of total 20-22 h, with pooling of the eluate for each cycle and column regeneration between total cycles. Each batch of pooled eluate was automatically adjusted to pH 3.4, held for 60 min, then automatically adjusted to pH 6.5, 10 mS/cm with 1 M Tris buffer and 1 M NaCl, and filtered with a micro filter with a nominal pore size of 0.1 µm (USP-043, Asahi Kasei Co., Ltd.) to remove precipitates. The process solution was supplied to the polishing process with two columns directly connected to a virus filter at a constant flow rate.
The total flow-through integrated polishing process consisted of two prepacked mini-columns of AEX (Cellufine MAX IB) and CEX (Cellufine MAX DexS-HbP; both Mini-Column 5LM; 14.6 mm ID, 30 mm L; CV of 5 mL; JNC Corporation) and a Planova BioEX virus filter (0.0003 m2; Asahi Kasei Medical) without pooling between each step, and the final purified mAb solution was collected. For the polishing chromatography steps, the AEX and CEX columns were directly connected in series to column valves of the AKTA pure 25 (Cytiva), equilibrated with 20 mM Tris-acetate, pH 6.5, 10 mS/cm, which was used for purification of mAb. The virus filter was connected to the outlet valve of the AKTA pure 25 and prepared for use independently of the columns. The AKTA pure 25 sample pump was operated at constant flow rate, and the virus filter inlet pressure (Figure 3) was recorded with a pressure sensor PREPS-N-000 (PendoTECH). Host cell proteins (HCP), host cell DNA and mAb concentrations and aggregates ratio were analyzed at each step of each batch by the following methods.
mAb concentration
mAb concentration (mg/mL) was determined in cell culture by analytical protein A-HPLC performed on a chromatography system consisting of MU701 UV-VIS detector and a PU7710 pump ( GL Sciences, Inc.) using a silica monolith Protein A column (Kyoto Monotech, Co., Ltd.) and for process solutions by bind and elute chromatography mode using 50 mM phosphate buffer with 1 M NaCl, pH 7.0 as the mobile wash phase and 100 mM sodium citrate, pH 3.5 as the mobile elution phase followed by quantification of mAb in purified solution by absorption measurement at 280 nm on a UV-VIS spectrophotometer V-750 (JASCO Corporation).