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).