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.