The Disposable Set Up
Described here is an experimental approach to improve the purification of MGG from serum at a 200-L production scale. The
objective was to purify mouse MGG with an integrated disposable assembly at a 3% scale that balanced availability of feedstock,
and to provide an appropriate scale of operation to accurately assess the scaled-down operation.
Materials and Methods
Figure 1
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The integrated disposable assembly (Figure 1) was built using neoprene tubing (Pharmed, Miami, FL) of suitable lengths and
with the appropriate connectors attached. These were placed in autoclave bags and sterilized at 121 °C for 20 minutes. The
filters were aseptically connected to tubing assemblies in a laminar flow hood.
Chromatography buffers were sterile filtered in bags using 0.2 μm filters (Opticap XL3) with Millipore Express SHF membrane
in the following quantities: equilibration buffer: 50 L; elution buffer: 10 L; and regeneration buffer: 50 L. The bags were
aseptically connected to the assembly as shown in Figure 1.
A 32 x 250 mm column (Millipore Vantage, Billerica, MA) was manually packed offline with chromatography media (ProSep-vA Ultra,
200 mL) in 20% benzyl alcohol. Packing linear flow velocities were 1,000 cm/hr. Before applying the feedstock, the column
was washed with 10 CV of elution buffer, followed by 10 CV of binding buffer. The column was then aseptically connected to
the disposable assembly as shown in Figure 1.
The TFF system was assembled, and the 30 kD membrane (Pellicon 3, 0.11 m2 ) was rinsed with 10 L of sterile water (Milli-Q) to remove any preservatives. The system was sanitized with 5 L of 0.5 M
NaOH. After sanitization, the assembly was rinsed with sterile water until a neutral effluent pH was measured from the cross
flow and permeate lines. The assembly was then aseptically connected to the eluate bag as shown in Figure 1.
Frozen mouse serum (6 L) was thawed then pumped into the disposable assembly. The flow rate was slowly ramped up to an inlet
pressure of 10 psi. All of the serum was allowed to pass first through the clarification filter (Opticap XL5 capsule with
Polysep II 1.0/0.2 μm cartridge), followed by the sterile filter (Millipak 200 gamma gold 0.22 μm filter) into a 10-L bag.
The clarification filter was changed twice because of by-plugging.
The serum was processed in a continuous operation in the assembly. A volume of 800 mL was pumped onto the column (ProSep)
at a flow rate of 60 mL/minute. The void volume for the tubing and column was found to be approximately 100 mL. The column
was washed with equilibration buffer (10 CV) at a flow rate of 120 mL/minute. The bound MGG was eluted with 3 CV of elution
buffer at a flow rate of 60 mL/minute. The pH of the eluate was neutralized by aseptic introduction of the neutralization
buffer. The column was regenerated with 10 CV of regeneration buffer and re-equilibrated with 10 CV of equilibration buffer.
Regeneration and re-equilibration were both done at a flow rate of 120 mL/minute. These steps were repeated eight times to
process the entire 6-L batch. The total volume used for neutralization of 6 L of eluate was 15 mL.
The purified MGG collected in the eluate bag was pumped into the TFF assembly. The polyclonal MGG was concentrated 16X and
constant volume diafiltered with PBS 0.01 M, NaCl 1.5 M, pH 7.4.
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