Currently, the benefits of membrane adsorbers are most apparent in flow-through applications because capacity constraints
in retention mode, particularly at high loading rates, make resin chromatography a more attractive option for capture steps.
Packed-bed chromatography certainly remains the preferred operation for capturing molecules of <200 kDa, especially when peak
cutting and gradients are required for the separation of closely related species.8 However, for larger molecules (including most of the anticipated contaminants in antibody manufacture), membranes offer
higher capacity and faster processing. For example, flow-through AEX for antibody polishing with a membrane adsorber can be
conducted with a bed height of 4 mm at flow rates of more than 600 cm/h, providing a much higher frontal surface area to bed
height ratio than is possible with columns. Even though the flow rate is much greater than would be possible with a column,
there is sufficient retention time to reduce DNA, most HCP, and many viruses by up to four log reduction values, allowing
membrane adsorbers to be used not only to separate the product from inert impurities but also as an integrated viral clearance
This article presents a new process developed at Philogen that uses membrane adsorbers for both flow-through and retention
steps in antibody polishing. The set-up was used successfully with Teleukin, a new MAb fusion protein in Phase 1–2 clinical
development at Philogen.11 After an initial capture step using a traditional Protein A column, the feed is loaded onto a Sartobind
Q AEX membrane adsorber (Sartorius Stedim Biotech, Göttingen, Germany), which elutes directly into a Sartobind S CEX adsorber
(Sartorius Stedim). The AEX adsorber is operated in flow-through mode to retain HCP, DNA, and leachate, while the CEX adsorber
operates in retention mode, allowing the pure antibody to be separated from positively charged impurities. Because the eluate
from the first adsorber is loaded directly and automatically onto the second, this can be regarded as a single, integrated
polishing step that achieves up to 90% recovery and 99.9% purity as determined by size exclusion chromatography, cation exchange
HPLC, and SDS–PAGE.
AFFINITY CHROMATOGRAPHY STEP
The initial step in the purification train is a standard Protein A affinity chromatography separation carried out by loading
the filtered culture medium from the bioreactor onto a column pre-equilibrated with phosphate-buffered saline (PBS). The medium
is loaded overnight at room temperature under conditions that allow a contact time of approximately 30 s. The column is then
washed through with an acetate saline buffer at pH 5.0, and the antibody is eluted with an acetate saline buffer at pH 3.0.
The eluted product is adjusted to pH 6.0 and diluted to a final conductivity of 8.0 mSi/cm before loading onto the Q/S dual
adsorber. As shown by the elution profile in Figure 1, it is possible to recover up to 90% of the product in this step at
a >90% purity.
Figure 1. Teleukin elution profile after the affinity chromatography step, based on absorbance at 280 nm