Purification of IgM Monoclonal Antibodies - Manufacturing challenges surround the use of IgM monoclonal - BioPharm International

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Purification of IgM Monoclonal Antibodies
Manufacturing challenges surround the use of IgM monoclonal


BioPharm International Supplements


In addition, the emerging generation of industrial ion exchangers includes solid phase supports that do not rely on diffusion. Both membranes and monoliths exploit convection for mass transport, and both have been applied effectively to IgM purification.15,19–21 Convection is independent of molecular size and flow rate, so dynamic binding capacity and resolution are unaffected by either parameter.22,23 Convective supports thereby offer a solution to the productivity bottleneck that afflicts traditional chromatography media. This solution has already had a positive impact with industrial purification of IgG, where anion exchange membranes are increasingly exploited for flow-through removal of host cell protein, DNA, endotoxin, and virus.24,25


Table 2. Initial screening conditions for hydroxyapatite
Membranes support less effective peak separation in bind–elute applications because of dispersion in membrane housings and between membrane layers. This dispersion produces a high degree of peak spreading, which erodes resolution and dilutes the eluted proteins. Monoliths lack these dispersion zones, and they further lack the void volume that truncates separation efficiency on porous particle media. These efficiencies result in sharper elution peaks, with the practical benefits of higher resolution and higher eluted product concentration. Capacities for large molecules are very high: dynamic binding capacity for endotoxin is more than 10 times higher on monoliths than on porous particle anion exchangers, and DNA capacity is nearly 50 times higher.26 These features all favor highly effective purification of IgM.

Materials and Methods


Table 3. Initial screening conditions for ion exchange
IgM cell culture supernatants (CCS) were filtered to 0.22 μm and stored at 4 °C. Buffer components were obtained from Sigma-Aldrich (St. Louis, MO). Buffers were made with water for injection (WFI) and filtered to 0.22 μm before use. Ceramic hydroxyapatite (CHT) Type II, 40 μm, was obtained from Bio-Rad Laboratories (Hercules, CA) and packed by Atoll GmbH (Weingarten, Germany) into 1 mL (5 x 50 mm) and 10 mL (11.3 x 100 mm) MediaScout columns. Monolithic convection interaction media (CIM) QA (quaternary amino) anion exchangers and CIM SO3 cation exchangers were obtained from BIA Separations GmbH (Klagenfurt, Austria). For initial screening and method development, 0.34 mL monolithic disks were used; 8 mL radial flow monoliths were used for process modeling. One mL RESOURCE ETH (ether) and PHE (phenyl) columns for HIC were obtained from GE Healthcare (Piscataway, NJ).


Table 4. Initial screening conditions for hydrophobic interaction chromatography
An experimental large-pore poly-propylene glycol (PPG) HIC resin from the Tosoh Resin Innovation Program (TRIP) was provided by Tosoh Bioscience (Montgomeryville, PA). It was packed in a 1 mL (5 x 50 mm) column. Screening buffers and conditions for hydroxyapatite are described in Table 2, for anion exchange and cation exchange in Table 3, and for HIC in Table 4. Dynamic binding capacity was determined as described in Gagnon.7 IgM breakthrough was detected with the monolith-based anion exchange assay described in Gagnon, Richieri, Zaidi, et al.27 All chromatography experiments were performed on an ÄKTAexplorer 100 from GE Healthcare. Polyacrylamide gel electrophoresis (PAGE) was performed on Bio-Rad Criterion gels (10–20% gradient).


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