March 15, 2012 (Vol. 32, No. 6)

Membrane Adsorbers Provide Alternative to Column Chromatography to Accelerate Purification

Membrane adsorbers accelerate purification of biotherapeutics due to their ease of use, small footprint, and flow rate independent binding. This tutorial examines the impurity removal offered by QyuSpeed™ D (QSD), a hollow fiber anion-exchange membrane adsorber developed by Asahi Kasei Medical.

QSD uses positively charged ligands elevated from the membrane surface by chains grafted on a 0.2 μm polyethylene membrane to remove negatively charged impurities including DNA, host cell proteins, and viruses in flow-through mode.

Application: Impurity Removal from Clarified Cell Culture

Initial experiments sought to test the hypothesis that QSD could be used in flow-through mode following cell culture clarification to remove impurities during one quick in-line step.

A 0.5 g/L model IgG cell culture solution at a pH of 7.5 and conductivity of 9.8 ms/cm was filtered through QSD and three other anion-exchange membranes to test this notion. After the cell mass was removed using a 0.45 μm polysulfone hollow fiber microfilter in tangential flow mode 100 membrane volumes (MV) of clarified cell culture solution was applied to each membrane at a flow rate of 5 MV/min.

Host cell protein (HCP) and DNA concentrations in the flow through material of each membrane were measured using ELISA (HCP) and the Quant-iT™ dsDNA BR assay kit (DNA) from Invitrogen.

As Figure 1 illustrates, QSD successfully removed contaminants from clarified cell culture. The pH (7.5) and conductivity (9.8 ms/cm) of the cell culture solution proved to be in the optimal range for QSD.

Excellent removal of contaminants coupled with flexible flow rates intrinsic to membrane adsorbers suggests that QSD is a robust tool for use post-clarification. The efficiency at which impurities are eliminated reduces the load on purification steps downstream of QSD while the flow rate independent binding and salt tolerance support in-line compatibility of QSD with a microfilter or other clarification steps without the need for solution adjustment.

Figure 1. Concentration of HCP (A) and DNA (B) in the load material and flow-through pools of membranes A, B, C, and QSD.

Application: Polishing Step Impurity Removal

The high level of impurity binding suggests a role for QSD as an alternative to traditional column chromatography during polishing. To assess the potential of QSD as a polishing step, HCP and DNA removal was performed using CHO cell culture clarified first using a 0.45 μm polysulfone microfilter in tangential flow mode, followed by a 0.2 μm sterilizing-grade filter.

Host cell proteins in solution were concentrated 4X using an ultrafiltration (UF) membrane and buffer exchanged 3X into the desired buffer solution. Test materials were diluted with the appropriate buffer to obtain an HCP protein concentration representative of a monoclonal antibody (mAb) polishing step (0.1 g HCP/L).

The HCP solution was subsequently filtered in flow-through mode through QSD and membrane B at a flow rate of 13.3 MV/min to a throughput of 30 g HCP/L-adsorbent. DNA and HCP were quantified using ELISA and the Quant-iT™ dsDNA DR kit (Invitrogen), respectively.

Figure 2. Percentage of DNA retained by QSD and membrane B in phosphate buffer at pH 7.5.

QSD removed DNA across a range of salt concentrations due to the unique grafted chain structure and DEA ligands. In contrast, in Figure 2, membrane B displayed a sharp decrease in its ability to remove DNA in the presence of 0.3 M NaCl.

Salt is a common component of many solutions and the removal of HCP in the presence of sodium chloride is summarized in Figure 3. In the absence of sodium chloride, membrane B showed higher HCP removal. However, as the salt concentration increased, the performance of membrane B decreased sharply while QSD maintained its HCP removal capability. Figure 4 demonstrates how QSD removed HCP across a range of buffers (all 50 mM), pH levels, and NaCl concentrations.

Figure 3. Percentage of HCP retained by QSD and membrane B in phosphate buffer at pH 7.5.

As the salt concentration increased to 0.3 M, the type of buffer greatly impacted the ability of QSD to bind HCP. At high salt concentrations, the optimal buffer conditions for QSD were determined to be phosphate with a pH of 7.5 and acetate with pH of 6.0. Therefore, QSD possesses excellent functionality in a phosphate buffer across a range of salt concentrations.

The abilities of QSD, membrane A, and membrane B to remove viruses were evaluated when a 0.5% v/v serum-free porcine parvovirus (PPV) spike was loaded in 10 g/L h-IgG, 0.1 M NaCl pH 7.9 solution. All membranes were operated in flow-through mode at a flow rate of 5 MV/min.

Figure 4. Percentage of HCP retained by QSD in a variety of buffering solutions between pH 6 and pH 8.5.

The log reduction value (LRV) evaluation was performed using the hemagglutination TCID50 assay. Compared to membranes A and B in Figure 5, QSD demonstrated a much higher capacity for virus removal. QSD showed complete PPV clearance out to 1,250 membrane volumes (12.5 kg h-IgG/L-adsorbent), while membranes A and B only showed complete clearance out to 291 membrane volumes and 222 membrane volumes, respectively.

Figure 5. PPV Clearance for flow-through fractions of QSD, membrane A and membrane B.


QyuSpeed D successfully removed impurities and demonstrated compatibility with buffers commonly used during purification. Cell culture solutions filtered through QSD did not require solution adjustment to achieve impurity reduction. Thus, QSD is a scalable, safe, and robust alternative to column chromatography for purification of biotherapeutic products and plasma derivatives.

Michael Thomas ([email protected]) is a field application scientist at Asahi Kasei Bioprocess.

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