Separation of IgM using TSK-GEL BioAssist S Columns

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Separation of IgM using TSK-GEL BioAssist S Columns

T.J. Higley, Tosoh Bioscience LLC

ABSTRACT:

A novel macroporous ion exchange material was developed to provide high retention and capacity of large biomolecules such as IgM. TSKgel BioAssist columns are offered in an analytical format packed with 7 micron particles and, most recently, in a semi-preparative format packed with 13 micron particles.

INTRODUCTION:

The use of monoclonal antibodies (mAb) is becoming more important in diagnosis and treatment of disease. Relative to other immunoglobulin classes, IgM has shown to possess unique and beneficial characteristics.

IgM is a very large molecule comprised of five IgG subunits resulting in a relatively labile and difficult-to-purify molecule. Unlike single chain antibodies, IgM, because of its size, cannot be purified by Protein A, an affinity material commonly used for its high binding capacity and excellent selectivity for antibodies. Alternative affinity methods have been developed with thiophillic adsorbents but often result in low binding capacity for IgG.

In this note, we examine an alternative purification method of IgM by ion exchange chromatography using a novel high capacity stationary phase. Additionally, scalability data is shared that documents consistent results from the analytical column to the recently commercialized 13 micron semi-preparative column.

CONDITIONS:

Cation Exchange Separation Method: (Figures 1 and 3)

Column:	TSKgel BioAssist S, 7 μm 4.6 mmID × 5 cm PEEK Housing
Eluent:	20 mM sodium phosphate buffer pH 6.0
Gradient:	step from 0 --> 0.3 M NaCl at t=5 min., and 0.3 M --> 0.5 M NaCl at t=10 min.
Flow Rate:	1 mL/min
Sample:	500 μL of 9.5 mg/mL IgM in mouse ascites fluid; shaded peaks represent albumin and IgM, respectively
Detection:	UV 280nm
Instrument:	Tosoh HPLC

Purity Determination by Size Exclusion Chromatography: (Figure 2)

Column:	TSKgel BioAssist G4SW_XL, 8 μmID × 30 cm, PEEK Housing
Eluent:	0.3 M NaCl in 50 mM sodium phosphate buffer pH 6.5
Flow Rate:	1 mL/min
Sample:	Fractions 1 and 2 from Cation Exchange Purification
Detection:	UV 280 nm
Instrument:	Tosoh HPLC

RESULTS:

Efficient and high-capacity separations of macromolecules such as IgM require small, macroporous particles. TSKgel BioAssist S columns are packed with spherical 7-micron particles that have an average pore size of 1300Aring>. As shown in Figure 1, baseline separation of IgM from other contaminants on TSKgel BioAssist S is achieved using a 0.3 M NaCl step gradient after elution of albumin. Linear gradient separations, although possible, did not yield equivalent baseline resolution (data not shown). Purity analysis by size exclusion of both fractions taken during cation exchange is shown in Figure 2. The IgM fraction was determined to be 97% pure. Interestingly, our work showed that increasing the sample load increased the purity of the IgM fraction. Figure 3 shows that below 0.75 mg loading, the purity decreases rapidly. It is postulated that IgM competitively displaces albumin from the sulfopropyl binding sites accounting for the increase in purity with increasing IgM concentration.

The separation performance is maintained up to a capacity of over 60 mg IgM per mL of resin (data not shown), or approximately 50 mg per 4.6mm ID × 5cm column. Researchers with a need to purify amounts greater than 50 mg/column, should use the recently commercialized 10 mm ID × 10 cm semi-preparative column, which is packed with 13 micron particles. To compensate for the loss in resolution attributed to the increase in particle size, the semi-preparative column was lengthened relative to the analytical format.

CONCLUSIONS:

The macroporous TSKgel BioAssist S column offers high binding capacity and excellent resolution for purifications of IgM and IgG antibodies. More information about column attributes or the IgM purification are available by requesting Separation Report SR100 or Technical Presentation P100 respectively at info.sep.am@tosohbioscience.com.

Tosoh Bioscience LLC
156 Keystone Drive
Montgomeryville, PA 18936
www.tosohbioscience.com

Tosoh Bioscience LLC
156 Keystone Dr.
Montgomeryville, PA, 18936

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