A New Scale-Down Membrane Adsorber Device for Process Development and Validation - The authors describe the development of an ultra scale-down anion exchange membrane adsorber, and demonstrate scalabi
Figure 3: Normalized flow (MV/min) for Sartobind pico and nano devices as a function of inlet pressure. MV is membrane volume.
Device geometry must allow for linear scalability through the entire device size range. Pressure flow curves were generated
with the axial flow Sartobind pico and radial flow Sartobind nano devices with data shown in Figure 3. The normalized flow rate (membrane volume (MV)/minute) increased linearly with the increasing inlet pressure and the flow
rates were comparable, suggesting effective flow distribution and efficient utilization of membrane area with both pico and
nano devices.
Figure 4: Filtrate flow rate at 3 bar constant initial pressure during loading of 20 g/L γ-globulin protein solution. MV is
membrane volume.
For a typical polishing application with an AEX membrane adsorber, the load capacity is very high, exceeding 10 kg of protein
feedstock per liter of membrane volume and can thus present the risk of membrane fouling. To assess fouling as a function
of load capacity, the Sartobind pico and Sartobind nano devices were loaded with a 20 g/L γ-globulin solution to a load capacity
of 20 kg/L at a constant inlet pressure of 3 bar. As seen in Figure 4, while slightly higher flow decay was observed with the pico device, the overall flow decay was minimal with the two devices
thus demonstrating the absence of significant membrane fouling at high load density.
Characterization of membrane adsorber devices using model systems
Figure 5: Bovine serum albumin breakthrough curves for pico, nano, and 10" devices. MV is membrane volume.
Chromatography media are usually characterized using model molecules, with dynamic binding capacity and impurity clearance
reported at specific process conditions. The dynamic binding capacity for Sartobind STIC-PA was determined using bovine serum
albumin (BSA) and DNA, and impurity clearance was evaluated using DNA, endotoxin, and bacteriophage.
Figure 6: DNA breakthrough curves for pico, nano, and 10" devices. MV is membrane volume.
Dynamic binding capacity: The dynamic binding capacity at 10% breakthrough was measured for the Sartobind pico, the Sartobind nano, and the Sartobind
SingleSep 10" capsule using BSA and DNA model systems. All devices were assembled with STIC-PA membranes. The breakthrough
curves for the three devices are shown in Figures 5 and 6 for BSA and DNA, respectively. The breakthrough curves are similar for all devices suggesting consistent flow distribution
and efficient utilization of the membrane binding sites at the three scales. Table II shows the average BSA and DNA dynamic binding capacity values for several Sartobind pico, nano and 10" devices. At 10% breakthrough,
the difference in dynamic binding capacity for all three devices was insignificant. The consistent dynamic binding capacity
with BSA and DNA supports a linear scalability from 0.08 mL axial flow pico device to 180 mL radial flow SingleSep 10" capsule.
Table II: Dynamic binding capacity (DBC) at 10% breakthrough using bovine serum albumin (BSA) and DNA model molecules. BSA
is bovine serum albumin.
Removal of bacteriophage: Pathogen clearance was evaluated using the bacteriophage ΦX174, serving as a surrogate for mouse minute virus (MMV), which
is typically used as a model virus for virus validation studies. Both ΦX174 (26-33 nm diameter) and MMV (20 nm diameter) are
small nonenveloped DNA viruses with an isoelectric point of around 6.7–7.0 and 6.2 respectively (13). At pH > 7, both ΦX174
and MMV are mainly negatively charged and expected to bind to positively charged AEX chromatography membranes, resulting in
their clearance from protein feedstock through electrostatic interactions. To compare clearance between Sartobind pico and
Sartobind nano, the same ratio of ΦX174 to membrane volume was loaded. Process-scale capsules were not tested because of the
large amount of phage material required. Two flow-through fractions were collected with each pico and nano device, and the
LRV was evaluated by comparing the phage titers of the fractions with the load solution. As shown in Table III, similar LRVs were obtained at a load of 100 and 150 MV of phage-spiked buffer, demonstrating linear scalability between
the devices.
Table III: Log reduction value of bacteriophage φX174 with Sartobind pico and nano devices. MV is membrane volume.
Removal of endotoxin: Endotoxins are lipopolysaccharides found in the outer membrane of various gram negative bacteria, can be present as different
forms of micelles and vesicles, and are generally strongly negatively charged. Because of their ability to elicit immunogenic
responses in humans, endotoxins must be removed to typically < 0.25 Endotoxin Units per milliliter (EU/mL) where EU is the
unit of measurement for endotoxin activity (USP <29>). Table IV shows the results for endotoxin removal with Sartobind pico and nano devices at pH 7.3 in a buffer containing 150 mM sodium
chloride. The concentration of endotoxin in the load was 108 EU/mL, which is significantly higher than the concentration of
endotoxin typically found in any in-process pools. Three fractions were collected from the flow-through at loading volumes
of 50, 100, and 150 MV. All flow-through fractions had an endotoxin concentration below the detection limit of 0.012 EU/mL
resulting in a LRV > 3.96 except one fraction at 50 MV with the pico device. However, subsequent fractions at higher load
volumes with the same pico device provided LRV > 3.96 which suggests that the anomalous reading at 50 MV was likely due to
an assay error or sample contamination. Based on the load volumes tested, the total amount of endotoxin removal was > 1296
EU with the pico and > 16200 EU with the nano device. Significantly larger amount of endotoxin would be required in the load
to saturate the membrane with the endotoxin molecules to determine and compare the breakthrough curves for both pico and nano
devices.
Performance of Sartobind pico with an industrially relevant mAb feedstream
Table IV: Endotoxin removal (log reduction value) at pH 7.3 in buffer containing 150 mM NaCl with Sartobind pico and nano
devices. MV is membrane volume.
In a mAb purification process, AEX chromatography is typically operated in a flow-through mode to bind trace levels of impurities
such as DNA, putative viruses, endotoxins, and host cell proteins, while the mAb product flows through. The load capacity
is indicated as the mass of product loaded per unit volume of chromatography membrane (kg mAb/L membrane) such that the purity
level in the product pool is acceptable. To assess the performance with an industrially relevant feedstream, both pico and
nano devices were loaded with an in-process mAb pool post Protein A and cation exchange chromatography step. Subsequently,
CHOP levels were monitored in the flow-through as a function of load density. The devices were loaded to 10 kg/L load density
at two different solution conditions (pH 7.0 and 8.0 at 11 mS/cm). CHOP clearance as a function of load density is shown in
Figure 7. Comparable CHOP clearance was obtained with the pico and the nano device at both solution conditions using an industrially
relevant mAb feedstock, suggesting that the Sartobind pico is scalable to the Sartobind nano device. Additionally, at pH 7.0
and 11 mS/cm, a load capacity ≥ 10 kg/L could be achieved with pool CHOP levels < 10 ppm.
Figure 7: Chinese hamster ovary proteins (CHOP) breakthrough curves for Sartobind pico and nano with a mAb feedstream. MAb
feedstock contained 100 ppm CHOP. Experiments were performed at pH 8.0 and 7.0 at 11 mS/cm and at a flow rate of 10 MV/min.
The CHOP clearance results are consistent with the earlier data where comparable BSA and DNA dynamic binding capacity was
observed between the pico, nano, and process scale 10" devices. Comparable clearance of endotoxin and the bacteriophage further
demonstrated the scalability of Sartobind pico to the Sartobind nano.
Nathalie Frau, PhD, is a senior scientist in purification process development, biotechnology division, Sartorius Stedim North America.
Articles by Nathalie Frau, PhD
