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Mar 15, 2013 (Vol. 33, No. 6)

Steric Exclusion Chromatography

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    Figure 1. Fundamentals of steric exclusion chromatography: The first panel shows proteins suspended in an aqueous buffer in the presence of a hydrophilic chromatography surface. The second panel shows the formation of PEG-deficient zones created by steric exclusion of PEG from hydrophilic surfaces. The third panel illustrates the reduction of interfacial area between the high-PEG bulk solvent and the PEG-deficient zones. This reduces free energy in the system. Excess water is released to the bulk solvent (fourth panel), reducing the bulk PEG concentration, which reduces the discontinuity between the high-PEG bulk solvent and PEG-deficient zones, further reducing free energy in the system. The fifth and last panel shows the system at equilibrium with proteins eliminated from the bulk solvent. Elution is mediated by reducing the PEG concentration.

    Most adsorption chromatography methods are based on a direct chemical interaction of a solid-phase ligand with a complementary target site on some population of biomolecules.

    Examples include ion-exchange chromatography, where solid-phase charges interact electrostatically with oppositely charged residues on biomolecules; hydrophobic interaction chromatography, where solid-phase hydrophobic ligands interact with hydrophobic residues on biomolecules; and bioaffinity chromatography, where complex affinity ligands interact with specific biomolecule structures in a lock-and-key fashion.

    Researchers at the Bioprocessing Technology Institute in Singapore have discovered a method of achieving adsorption where there is no direct chemical interaction between the solid phase and the target biomolecule. Instead, a biomolecule is induced to associate with a solid phase by their mutual repulsion of a promoter substance.

    The technique is called steric exclusion chromatography (SXC) and it shares common mechanistic roots with the method of precipitation with polyethylene glycol (PEG, Figure 1). The main operational difference is that SXC employs a hydrophilic solid phase as a nucleation center on which biomolecules accrete instead of forming precipitates.

    The key performance differences are superfast binding kinetics, better purification, better product recovery, and better process control, all of which derive from the contribution of the solid phase. The technique can be performed in monolith and fluidized particle formats, but does not work in columns packed with porous particles. Selectivity is based on biomolecule size. SXC behaves like a rapid high-capacity alternative to size exclusion chromatography.

  • Purification of Virus Particles

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    Figure 2. Virus purification by steric exclusion chromatography [Modified from J. Lee et al., J. Chromatogr 2012]

    Virus particles are a difficult challenge for purification with columns of packed porous particles because such columns rely on diffusion for the virus particles to be transported into the pores where most of the binding surface area resides. The large size of these particles translates to extremely slow diffusion constants. The low efficiency of diffusion reduces binding capacity and resolution.

    Besides that, many species of virus are so large that they cannot fit into the pores at all, so binding capacity is further limited to the external surface of the particles. Capacity is frequently 1010 particles or less per mL of chromatography media. Figure 2 shows a chromatogram of bacteriophage M13 purified from E. coli broth by SXC on a hydroxyl monolith.

    Dynamic binding capacity was 1013 particles per mL, and the technique achieved 99.8% reduction of E. coli host proteins in a single step. DNA was reduced 93%. Despite the elevated viscosity due to the presence of PEG, more than 90% of the virus was captured during the 6 seconds transit time of a given unit of sample through the monolith. This corresponds to a flow rate of 10 bed volumes per minute that supports very short process times.

    Two additional benefits of the method are that PEG is virus-stabilizing, and flow-through monolith channels is laminar. The latter feature avoids the destructive shear forces that occur in the void volume of packed particle columns. As a result, full infectivity of the purified virus was conserved.

    The combination of these results makes SXC on monoliths thousands of times more effective and productive than chromatography on columns packed with porous particles. Similar results have since been achieved with several other bacteriophage species as well as influenza and Dengue virus. These results suggest that the technique will prove to be a universal method for virus purification.

    This brings up the issue of how to purify virus particles using SXC. This requires a binary gradient chromatography system with pressure capabilities of at least 2 MPa.

    Hydroxyl monoliths ranging from 1 mL to 8 L are available from BIA Separations. Purification of the M13 phage was done by equilibrating the A-pump inlet line with 12% PEG-6000, 500 mM NaCl, 50 mM MES, pH 6.0, and the B-pump inlet with 500 mM NaCl with, 50 mM MES, pH 6.0.

    The monolith was equilibrated with 50% A/50% B at 10-bed volumes per minute. The system was paused and the B-pump inlet transferred to the sample to be purified.

    When the sample was fully loaded, the system was paused and the pump-B inlet transferred back to the original buffer. The monolith was washed to baseline, and the virus eluted with a 10-bed volume linear gradient to 100% B.

  • Purification of IgG mAbs

    Protein A affinity chromatography currently dominates the field of IgG purification but SXC offers features that protein A cannot. This application employs single-use hydroxylated particles that cost less than $100/L, but offer binding capacities greater than 200 g/L.

    The method is performed in a fluidized bed format with the aid of tangential flow filtration. It requires no columns, and there is no scale limitation, so an entire cell culture production batch can be processed in a single cycle requiring less than an 8-hour shift.

    Early versions of the technique achieved reduction of host cell proteins to the range of 3,000 to 4,000 ppm, but recent improvements support reductions to less than 200 ppm. IgG recovery ranges from 90–95%.

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    Table. Cost Factors for IgG Capture

    From there, a final purification step achieves HCP levels below 1 ppm, and aggregate levels typically less than 0.1%. Most of the characterization work to date has been done with a Trastuzumab biosimilar produced in CHO cells at about 2 g/L, but Bevacizumab and Adalimumab biosimilars give equivalent results. Cost factors of SXC are compared with protein A in the Table.

    How to purify IgG monoclonal antibodies using SXC? This requires a tangential flow filtration system with 0.22 or 0.45 µm membranes. The technique also requires nonporous hydroxylated particles. It is anticipated that the current prototype average 30 µm SXC particles will become commercially available in 2013.

    For supernatants containing about 2 g/L of antibody, add particles to clarified supernatant in an amount of 4% (w/v). Adjust pH to about 8.0. The closer to the antibody isoelectric point, the lower the PEG concentration required to achieve binding.

    Begin with salt concentration at about physiological since additional salt has been shown to reduce the effectiveness of PEG. Add PEG-6000 to its final concentration. With anti-HER2, this is about 18%. Incubate stirring for 30 minutes. Replace the reaction buffer with clean PEG buffer by diafiltration. The antibody remains in the retentate by virtue of being associated with the particles.

    Reduce the suspension volume and introduce buffer lacking PEG to dissociate the IgG from the particles. Collect the IgG in the filtrate. Apply the filtrate to a second chromatography step where the antibody binds. Current results indicate this can be cation exchange (many vendors) or mixed-mode methods such as hydroxyapatite (Bio-Rad), Capto MMC or Capto adhere (GE Healthcare).

    The PEG flows through the column and is eliminated. Elute the purified antibody.

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