Hydrophobic Membrane Adsorbers for Large-Scale Downstream Processing

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BioPharm International, BioPharm International-10-03-2009, Volume 2009 Supplement, Issue 7

This alternative to column chromatography is suitable for flow-through as well as bind-and-elute purification operations.


Ion exchange–based membrane chromatography has already proven to be a powerful alternative to polishing columns in flow-through mode for contaminant removal. More recently, new membrane adsorbers have been designed for use in other applications. This article discusses a Sartobind phenyl membrane adsorber that has been developed for the manufacturing-scale production of biomolecules based on hydrophobic interaction chromatography (HIC) principles. The new adsorptive membrane combines the advantages of membrane chromatography with a high binding capacity for proteins comparable to that of conventional HIC resins. It represents a new tool for downstream processing, applicable for both bind-and-elute and flow-through operations.

In the past decade, there has been a trend in the biopharmaceutical industry toward higher upstream productivity, i.e., higher cell culture titers, to meet the ever-growing clinical and commercial demand.1 This trend poses a challenge for the industry—to develop and scale-up downstream purification processes capable of purifying multiple kilograms per batch. Although downstream processing faces significant bottlenecks because of heavy reliance on traditional bead-based chromatography.2,3 For example, although Protein A chromatography has undergone great optimization for throughput in the past five years, the expected rise in cell culture titers, combined with the need for larger masses of antibodies, indicates a need for further improvement of the existing Protein A resin to accommodate the demand for high-throughput production.4 Therefore, there has been significant interest in the development of new technologies that facilitate the processing of multikilogram batches at the lowest possible cost.2,5 Among the novel technologies developed, membrane chromatography has a high potential, particularly for process-scale monoclonal antibody (MAb) purification.

Sartorius Stedim Biotech

Membranes feature a more open structure than resins, so there is virtually no diffusion limitation, and as a result they offer many technical advantages that have been well described in the literature.6–9 Several applications have been discussed for MAb purification. Because of the generally high isoelectric points of MAbs, anion exchange (AEX) resins are typically used as a polishing step in a flow-through mode (i.e., the product does not bind while the trace impurities such as DNA, viruses, endotoxins, and host cell proteins are retained).10 Although the amount of impurities to bind is very low in such operations, conventional polishing scale-up leads to column oversizing because of the pressure and diffusion limitations associated with conventional bead-based chromatography. In contrast, a disposable membrane chromatography device has a convective mode of mass transport, which allows operation at significantly higher linear flow-rates (shorter residence times) than columns. As a result, disposable membrane chromatography device can have a much smaller volume than that of a conventional column when used in flow-through mode. This significantly reduces buffer consumption, processing time, and floor space requirements.11 Sartobind ion exchange capsules, for example, have been successfully implemented in various downstream processes for the removal of negatively charged contaminants.12–15 It is well documented in the literature that AEX membrane adsorbers are a powerful alternative to columns and can facilitate the development of new purification strategies for downstream processing.

In addition to positively charged membranes, hydrophobic interaction membrane chromatography (HIC) has been described for the purification of a humanized MAb.16 Hydrophobic interaction is listed among the most commonly used chromatography methods for protein purification. For MAb purification, HIC is also often operated in flow-through mode.10 It is used to separate molecules based on their difference in hydrophobicity, and the technique has been presented as an efficient mode to remove dimers and high molecular weight (HMW) aggregates as a polishing step in a MAb purification process.17 In this article, we describe the use of an HIC membrane adsorber for the purification of a monoclonal IgG1 expressed using the PER.C6 human cell line. The hydrophobic membrane adsorber is based on hydrophilic regenerated stabilized cellulose with hydrophobic phenyl groups covalently attached to the base matrix.

A Phenyl Membrane Adsorber for Large-Scale Purification

The Sartobind phenyl hydrophobic membrane adsorber was recently introduced to the market.18,19 It is assembled into 30-layer radial flow process capsules with an 8-mm bed height. The HIC membrane adsorber resulted from the combination of a newly designed macroporous membrane structure and covalently attached hydrophobic phenyl ligand. This membrane structure was designed for high flow rates and binding capacities. Furthermore, the hydrophilic stabilized regenerated cellulose membrane support possesses excellent mechanical and chemical robustness and demonstrates minimal unspecific interaction with proteins, even at high concentrations of a lyotropic salt.19 The functionalized membrane exhibits increasing binding capacity in increasing ammonium sulfate concentrations, as expected in HIC. Table 1 describes the binding capacity at 10% breakthrough for Sartobind phenyl challenged with three different model proteins.

Table 1. Dynamic binding capacities (DBC) at 10% breakthrough for Sartobind phenyl membrane adsorber (3 mL membrane volume) challenged with three different model proteins. All proteins were prepared as 1 g/L solutions in 50 mM phosphate buffer at pH 7.0 with varying amounts of ammonium sulfate, and loading was done at 10 mL/min (18 s residence time).

In a recent paper, the separation properties of the HIC membrane were evaluated and compared to those of a traditional HIC resin, phenyl Sepharose FF (low sub) from GE Healthcare (Chalfont St. Giles, UK).19 Using a protein mixture of cytochrome C, trypsinogen, and a polyclonal IgG, Sartobind phenyl demonstrated better resolution than the HIC resin under the experimental conditions used for the evaluation. In addition to the bind-and-elute operation, the authors demonstrated the application of Sartobind phenyl in flow-through mode for aggregate removal in a purification process for a recombinant protein. The protein was loaded with varying amounts of ammonium sulfate such that the aggregates bound while the monomer did not. After loading, the membrane was washed with deionized water to elute the bound aggregates. The aggregate removal increased with increased ammonium sulfate concentration, as shown in Figure 1. Protein yield was >95%, regardless of the salt conditions used in the loading buffer.


Figure 1. Flow-through mode operation with Sartobind phenyl membrane adsorber to remove high molecular weight (HMW) aggregates for the purification of an E. coli expressed protein. The figure shows the percentage of aggregates bound onto the membrane at increasing salt conditions.

A Case Study

In this case study, a human MAb (IgG1, pI = 8.3, 150 kDa) was purified using the Sartobind phenyl membrane adsorber in flow-through mode. The protein of interest was produced at Percivia, LLC, using the PER.C6 cell line in a fed-batch process with a chemically defined growth medium. PER.C6 cells are E1-immortalized human embryonic retinal cells described in US patent 5,994,128.20 The crude media was clarified by centrifugation at 15,000g followed by depth filtration and sterile filtration. The clarified media was partially purified by column chromatography.

This material was loaded onto a 3 mL Sartobind phenyl nano membrane adsorber in 50 mM sodium phosphate buffer, pH 7.0, with ammonium sulfate concentrations ranging from 0.1 to 0.4 M. The product was collected in flow-through fractions and the membrane was then washed with equilibration buffer to recover the entire product. Samples of the flow-through fractions were assayed for aggregates by size exclusion chromatography.

Figure 2. Normalized aggregate levels of flow-through fractions collected while loading Sartobind phenyl membrane adsorber with partially purified antibody at various ammonium sulfate concentrations. (HMW: high molecular weight)

Figure 2 shows that the binding of HMW aggregates improved with increased salt concentration in loading buffer. Although 100% breakthrough of aggregates was observed at 0.1 and 0.2 M (NH4)2SO4, saturation was not reached at 0.3 or 0.4 M (NH4)2SO4 when the membrane was loaded with up to 167 mg-MAb/mL-membrane. As shown in Figure 3, protein recovery was greater than 94%, even at the highest salt condition of 0.4 M (NH4)2SO4, while aggregate reduction for the total flow-through pool was approximately 50%. In this study, a loading capacity of at least 167 mg-MAb/mL-membrane was achieved using the Sartobind phenyl membrane adsorber with yields of ≥94% and a final aggregate level <1% for a monoclonal IgG1.

Figure 3. Yield and high molecular weight (HMW) aggregate reduction for Sartobind phenyl membrane adsorber loaded with partially purified antibody at various ammonium sulfate concentrations


Featuring high binding capacity comparable to that of conventional HIC resins even at significantly higher flow rates, Sartobind phenyl membrane adsorber represents a new membrane-based tool applicable for flow-through as well as bind-and-elute purification operations. It can be considered as an alternative to column chromatography in processes where throughput, binding capacity, or resolution need improvement.

In addition, replacing several hundred liter columns with prepacked disposable membrane adsorbers can provide other advantages—such as a smaller plant footprint, the elimination of column packing and subsequent cleaning validation, and significantly lower buffer volume requirements—while adding flexibility to the downstream process.

NATHALIE FRAUD, PhD, is a senior scientist in purification process development, biotechnology division, Sartorius Stedim North America, Bohemia, NY, 626.241.2171, nathalie.fraud@sartorius-stedim.com and MIYAKO HIRAI is a product manager of membrane chromatography, biotechnology division, Sartorius Stedim Biotech GmbH, Goettingen, Germany, MICHAEL KUCZEWSKI, PhD, is an associate scientist III and GREGORY ZARBIS-PAPASTOITSIS, PhD, is a director, both in the downstream process development department at Percivia LLC, Cambridge, MA.


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