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, email@example.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.
1. Wurm F. Production of recombinant protein therapeutics in cultivated mammalian cells. Nature Biotechnol. 2004;22:1–6.
2. Gottschalk U. Bioseparation in antibody manufacturing: the good, the bad and the ugly. Biotechnol Prog. 2008;24:496–503.
3. Scott S. Proactive debottlenecking. Planning ahead for the downstream bottleneck. BioProcess Int. 2008 Special Report;(21)9:2–7.
4. Low D, OLeary R, Pujar NS. Future of antibody purification. J Chromatogr B. 2007;848:48–63.
5. Thoemmes J, Etzel M. Alternatives to chromatographic separations. Biotechnol Prog. 2007;23:42–45.
6. Fraud N. Membrane chromatography: an alternative to polishing column chromatography. Bioprocessing J. 2008:7:34–37.
7. Ghosh R. Protein separation using membrane chromatography: opportunities and challenges. J Chromatogr A. 2002;952:13–27.
8. Lim JAC, Sinclair A, Kim DS, Gottschalk U. Economic benefits of single-use membrane chromatography in polishing. BioProcess
9. Mora J, Sinclair A, Delmdahl N, Gottschalk U. Disposable membrane chromatography. Performance analysis and economic cost
model. BioProcess Int. 2006(Suppl.);4:38–43.
10. Schukla A, Hubbard B, Tressel T, Guhan S, Low D. Downstream processing of monoclonal antibodies—application of platform
approaches. J Chromatogr B. 2007;848:28–39.
11. Zhou JX, Tressel T. Basic concepts in Q membrane chromatography for large-scale antibody production. Biotechnol Prog.
12. Zhou JX, Tressel T, Gottschalk U, Solamo F, Pastor A, Dermawan S, et al. New Q membrane scale-down model for process-scale
antibody purification. J Chromatogr A. 2006;1134:66–73.
13. Glynn J, Hagerty T, Pabst T, Annathur G, Thomas K, Johnson P, et al. The development and application of a monoclonal
antibody purification platform. Biopharm Int. Advancing in separation and purification: purifying high titers. 2009 Suppl;22(3):16–20.
14. Arunakumari A, Wang, Ferreira G. Improved downstream process design for human monoclonal antibody production. Biopharm
Intl. 2007 Suppl;20(10):6–10.
15. Arunakumari A, Wang J, Ferreira G. Advances in non-Protein A purification processes for human monoclonal antibodies.
Advancing in separation and purification: purifying high titers. Biopharm Int. Advances in process chromatography. 2009 Suppl;22(3):22–26.
16. Ghosh R, Wang, L. Purification of humanized monoclonal antibody by hydrophobic interaction membrane chromatography.
J Chromatogr A. 2006;1107:104–09.
17. Li F, Zhou JX, Yang X, Tressel T, Lee B. Current therapeutic antibody production and process optimization. Bioprocessing
18. Liu X, Colling M, Fraud N, Campbell J, Lowenstein I, Lacki KM, Rathore AS. Upcoming technologies to facilitate more
efficient biologics manufacturing. Biopharm Int. 2009:22(2);38–51.
19. Fraud N, Faber R, Kiss C, Demmer W, Hoerl HH, Fischer-Fruehholz S. Hydrophobic-interaction membrane chromatography for
large-scale purification of biopharmaceuticals. BioProcess Int. 2009 Suppl;22(7):30–5.
20. Fallaux F, Hoeben R, Van der Eb A, Bout A, Valerio D. IntroGene B.V., assignee. 1999; Nov 30. Packaging systems for
human recombinant adenovirus to be used in gene therapy patent 5994128.