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 1: A. Sartobind pico 0.08 mL. B. Sartobind nano 1 mL. (ALL FIGURES ARE COURTESY OF THE AUTHORS)
Sartobind pico, the new scale-down device was provided by Sartorius Stedim Biotech GmbH, Göttingen, Germany. The device consists
of 15 membrane layers with polypropylene sealing rings every 3 layers, and is assembled into a molded polypropylene housing
with luer lock connectors to enable easy connection to a liquid chromatography system (see Figures 1 and 2). The bed height of 4 mm is similar across the entire Sartobind SingleSep family and the frontal surface area of 20 mm2 gives pico a membrane volume of 0.08 mL. Sartobind nano, (Sartorius Stedim Biotech GmbH, Göttingen, Germany) with 15 layers,
36.4 cm2 total surface area, and 1 mL membrane volume was used as a reference device (see Figure 1). The Sartobind nano has a radial flow and is constructed in the same way as process scale SingleSep capsules, which assures
direct scalability to manufacturing scale capsules (7–11). The key attributes of Sartobind pico and Sartobind nano are summarized
in Table I.
Figure 2: Sartobind pico device design.
The Sartobind SingleSep 10" capsule with a membrane volume of 180 mL was used to further confirm scalability. The devices
were assembled with a salt tolerant AEX membrane, Sartobind STIC PA, consisting of a polyallylamine ligand covalently coupled
to the cellulose membrane matrix (12).
Equipment
Table I: Key attributes of Sartobind pico and Sartobind nano.
All laboratory-scale chromatography experiments with mAb feedstock, model proteins, and model DNA were performed using an
ÄKTA Explorer FPLC system (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA). The devices were connected to the ÄKTA
Explorer with standard tubing and luer-lock connectors. A flow rate of 10 membrane volume (MV)/min was used. Binding of endotoxin
and bacteriophage molecules was performed using a separate experimental setup consisting of a peristaltic pump (Watson Marlow
302S), which allowed proper cleaning of the system. To determine flow rates, membrane adsorber devices were connected to a
pressure vessel filled with buffer or protein solution. The filtrate volume was monitored using a balance and the flow rates
for different pressures were calculated up to an inlet pressure of 3 bar.
Model systems
Bovine serum albumin (BSA, Lot 50121326) was purchased from Kraeber GmbH & Co. and salmon sperm DNA (DNA, Lot 8087) from Biomol.
The protein throughput was determined using γ-globulin (Sigma, γ-globulin from bovine blood, Lot STB0227K9). Endotoxin from
Escherishia coli (Lonza LPS E. coli 055:B5 N185 Lot 0000100778) was used as standard. Bacteriophage ΦX174 (ATCC 13706-B1) was produced in a 50 L disposable bioreactor
using the E. coli (ATCC 13706) expression system. Subsequently, phage was purified, concentrated, and sterile filtered by several steps including
a depth filtration cascade, crossflow filtration, precipitation with polyethylene glycol, and centrifugation.
MAb feedstock
The mAb feedstock was obtained from pilot-scale batches produced at Genentech (a member of the Roche Group). It was expressed
in mammalian cells and clarified to remove insoluble impurities. The mAb was processed through a protein A chromatography
step and further purified using a cation-exchange chromatography step. Protein concentration was approximately 11 g/L.
Nathalie Frau, PhD, is a senior scientist in purification process development, biotechnology division, Sartorius Stedim North America.
Articles by Nathalie Frau, PhD
