Multimodal (mixed-mode) chromatography methods have existed since the 1950s (3). Hydroxyapatite (HA) is the archetype of multimodal chromatography methods, combining cation exchange and metal affinity. (4). It is also a prime example of the barriers that need to be overcome for mixed modes to become mainstream tools. HA's selectivity was recognized as unique from its introduction, but a lack of practical knowledge concerning its binding mechanisms long delayed the development of scouting pathways that fully revealed its abilities. That discouraged process developers who might have benefited from its capabilities. As those pathways were defined, it became possible to control each binding mechanism, and HA has emerged as the most broadly capable process option for removing fragments and high levels of aggregates from antibody preparations (5,6,7,8,9).

Successes in recombinant immunotherapy have stimulated introduction of other mixed-mode media. Charged-hydrophobic mixed modes began to appear in the 1980s, mostly as hopeful protein A replacements. They included products such as T-gel, ABx, Avid-AL, and MEP Hypercel media (10,11,12,13,14,15,16,17,18,19,20,21). More recent entries such as AcroSep HEA, AcroSep PPA, Capto MMC, and Capto adhere media have been applied for capture but focus more on aggregate removal (22,23,24,25,26).

Such products arrive at a propitious moment: HA's successes have made process developers willing to confront the complexity of mixed modes and provided a conceptual framework for exploring multimodal interactions. High-throughput screening and statistical design of experiments (DoE) enable rapid accumulation of process-pertinent data (7,25,26). And the well-defined chemical structures of mixed-mode ligands provide valuable guidance on the types of eluting agents that promise useful results (27).

Materials and Methods

Cell Culture: Anti-PSCA minibody (clone A11.1 2C3) was grown in NS0 cells using media supplemented with 2% fetal bovine serum. This minibody is about 80 kDa and has an isoelectric point (pI) of about 7.3. Initial purification process development was conducted with minibody produced in T flasks, and product concentration was ~50 mg/L. Later material produced in hollow-fiber bioreactors was at 1–2 g/L. All preparations contained a subpopulation of dimers created by noncovalent association of variable regions (28,29).

Media Conditioning: We obtained Dowex AG1x8 (cholestyramine) media — a particulate, microporous, strong anion exchanger on a hydrophobic styrene divinyl benzene backbone — from Bio-Rad Laboratories (www.bio-rad.com). It was added to harvested supernatant at a proportion of 5% (v/v) and incubated with gentle mixing overnight at 4 °C (29), then removed by membrane filtration at 0.22 µm. This method has been shown to remove cell debris, DNA, lipopolysaccharide, phospholipids, fatty acids, steroids, and pH indicator dyes (27), which constitute most of the primary foulants of chromatography media. It is effective even at physiological pH and conductivity levels, at which its affinity for IgG and minibodies is nil. Used media can be discarded.

Purification: All chromatography experiments were conducted using an ÄKTA 100 Explorer system from GE Healthcare (www.gelifesciences.com). Buffers and salts came from Sigma Chemical Company (www.sigmaaldrich.com) except for arginine from Ajinomoto Aminoscience (www.ajiaminoscience.com). Initial cation exchange (CX) and anion exchange (AX) scouting were conducted on 334-µL CIM SO₃ and CIM QA monoliths (12 mm diameter × 3 mm height) from BIA Separations (www.biaseparations.com).

These experiments were conducted at a linear flow rate of 300 cm/hr (4-mL/min volumetric flow rate). The CX sample was prepared by 5× dilution of filtered cell culture supernatant (CCS) with 20 mM MES at pH 6. The AX sample was prepared by 5× dilution of CCS with 20 mM Tris at pH 8. Initial scouting was performed with 5-min (60 column volume) conductivity gradients to 500 mM sodium chloride (NaCl). We used 20 mM MES for buffering at pH 6, 20 mM Hepes at pH 7, and 20 mM Tris at pH 8. CX experiments (pH 4.5) were conducted in 20 mM sodium acetate. Later-stage AX and CX applications were conducted with UNOsphere Q or UNOsphere S media from Bio-Rad at a 300-cm/h linear flow rate.

For hydrophobic-interaction chromatography (HIC) we used ToyoPearl Phenyl 600M from Tosoh BioScience (www.tosohbioscience.com) packed into an HR 5/5 column (5 × 50 mm) from GE Healthcare. Sample was prepared by 3× dilution of CCS with 4 M NaCl. The column was equilibrated with 20 mM sodium phosphate and 1.5 M ammonium sulfate at pH 7 and eluted with a linear gradient to 20 mM phosphate at pH 7.0. Linear flow rate was 300 cm/h (1 mL/min).

CHT type I ceramic hydroxyapatite (40 µm) from Bio-Rad was packed into a range of MediaScout Minichrom columns by ATOLL GmbH (www.atoll-bio.com). We ran experiments at a linear flow rate of 300 cm/h and applied CCS undiluted. For an initial scouting run, the column was equilibrated to 10 mM sodium phosphate at pH 6.5, eluted in a linear gradient to 250 mM phosphate, and cleaned with 500 mM phosphate. A second run was conducted by equilibrating to 20 mM sodium phosphate at pH 6.5, eluting with a linear gradient to 20 mM phosphate and 1 M NaCl, then cleaning with 500 mM phosphate.

We obtained Capto MMC media, 1-mL and 5-mL HiTrap columns, and bulk media from GE Healthcare and conducted scouting on a 1-mL HiTrap column (7 × 25 mm) at a linear flow rate of 150 cm/h (1 mL/min). Sample was prepared by 1:1 dilution of CCS with 50 mM MES at pH 6.0. The column was equilibrated with the same buffer, eluted with a linear gradient to 50 mM MES and 500 mM arginine, then cleaned with 2 M guanidine at pH 5.5. We used the higher MES concentration to compensate for buffer capacity of the MMC carboxyl group.

Analysis: We ran reduced and nonreduced sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on precast Ready Gel 10% TrisHC1 Ready gels from Bio-Rad. We calibrated protein size using SeeBlue Plus2 prestained standards from Invitrogen (www.invitrogen.com) and detected sample proteins by either Bio-Safe Coomassie stain from Bio-Rad or Western blot with affinity-purified goat antihuman Fc polyclonal antibody from Jackson Immunoresearch Laboratories (www.jacksonimmuno.com). Analytical size-exclusion chromatography (SEC) was run on Superdex 75 HR 10/30 columns (10 × 300 mm) from GE Healthcare in PBS from Irvine Scientific (www.irvinesci.com) at a linear flow rate of 37.5 cm/h (0.5 mL/min). We calibrated protein size using gel filtration molecular weight standards from Bio-Rad.