First-pass scouting results were revealing but disappointing. The minibody eluted from AX at about physiological conductivity (15.6 mS/cm) at pH 8 but only about 6 mS at pH 7.0. It eluted slightly in advance of bovine serum albumin (BSA) with small injections and extended linear gradients but as a leading shoulder on the BSA peak with larger loads. Some BSA bound at pH 6.0, but the minibody did not. It bound very strongly to CX at pH 4.5, requiring 48 mS/cm conductivity for elution, which corresponds to ~0.5 M NaCl. Binding was much weaker at pH 6.0, with the minibody eluting at ~9 mS/cm. It eluted slightly after BSA with small injections but as a trailing shoulder with larger column loads. Both the minibody and most BSA failed to bind CX at pH 7.0. HIC supported slightly better separation than AX or CX, but with BSA still trailing through the later-eluting minibody peak.

HA with phosphate gradients has been used successfully for purification of Fab (30), anticarcinoembryonic antigen diabody and minibody (29), but the anti-PSCA minibody coeluted on center with BSA (Figure 3). More recent work with HA describing the relative behavior of IgG, fragments, and BSA has shown that although BSA and Fc binding are dominated by calcium affinity, HA binding of Fab is dominated by cation exchange (31,32). This suggested that NaCl gradient elution might support more effective BSA removal, which it did (Figure 4). This was the first “eureka moment” in the development process, not only for BSA removal, but also because such gradients have been shown to remove >3 logs of DNA, >4 logs of endotoxin, and 4 logs of murine leukemia virus from IgG preparations (8,33). Chloride gradients typically support the most effective removal of IgG aggregates (8). Indeed, when we ran an extended postgradient hold at 1.0 M NaCl, a second product peak eluted (Figure 5) predominantly populated by product dimer.

This is unattractive in a capture step because initial sample composition is typically the most variable feature of a purification process. Variations are common in product concentration and the product/contaminant ratio. If such variation ripples through the capture step, it can affect the performance of subsequent purification steps and quality of a final product. We therefore evaluated a series of wash conditions in the hope of improving both purity and reproducibility of the MMC elution.

These experiments were conducted with 50-mL loads of diluted CCS to conserve sample, and indeed we developed conditions that increased purity up to ~75%. However, when the load increased to 180 mL of diluted CCS, most of the minibody eluted in the wash. This was even more undesirable than the original problem because it showed that not only binding selectivity, but also elution selectivity depended on column loading. That left us with the sole option of setting load specifications based on minibody concentration in the CCS — with the inherent risk that purification performance could still vary with the product/contaminant ratio. Downstream purification steps would need to accommodate this variation.

Given that an unoptimized scouting run on HA had already demonstrated the highest single-step purification potential, it seemed an obvious place to seek further improvements. We evaluated the effects of different phosphate concentrations on binding selectivity. Contaminant binding diminished progressively up to 25 mM phosphate. Minibody began to break through at higher concentrations. Previous experience has shown that HA binding capacity is inversely proportional to phosphate concentration ≥5 mM, which is required to maintain the stability of HA itself (6,34). We also determined that the most effective removal of contaminants occurred in a NaCl gradient conducted at 10 mM phosphate.

Accordingly, we equilibrated the sample by adding phosphate to a final concentration of 5 mM. The column was initially equilibrated with 25 mM phosphate at pH 7. Sample application reequilibrated the column to 5 mM phosphate for maximum binding capacity. Washing with 25 mM phosphate removed a suite of minor contaminants, including transferrin. Phosphate concentration was then reduced to 10 mM, and the column was eluted with a gradient to 1.0 M NaCl (10 mM phosphate at pH 7). The minibody eluted at ~800 mM NaCl. This sequence reduced buffer volume and process time from the more conventional approach — which would have required equilibration, loading, and a first wash, all at 5 mM phosphate, followed by a wash with 25 mM phosphate, then reequilibration to 10 mM phosphate before elution.

We added an AX step to ensure adequate virus removal. We used monoliths to scout and model conditions because they produce data much faster than other formats (<10 minutes from one run to the next rather than ~45 min for conventional media). Monoliths support ~1.5 logs higher DNA capacity and 2 logs higher virus capacity than porous particle media and have twice the capacity of membrane anion exchangers (35,36). Had AX been the final step in our process, we would likely have continued with monoliths, but that would have required a diafiltration step following the high-salt HA elution.

We chose instead to place AX before HA, which created a different compromise. Monoliths have relatively low binding capacity for small proteins, which probably would have been stressed by the ~50% BSA load coming from the MMC step. Competition from bound BSA might in turn have compromised the efficiency of DNA and virus removal. We therefore decided to use a high-capacity porous-particle anion exchanger and chose UNOsphere Q media, with a dynamic binding capacity of ~180 mg/mL BSA (37).

PHASE 1 PURIFICATION PROCESS

Capture on MMC: Dilute filtered supernatant 1:1 with 50 mM MES at pH 6. Equlibrate column with 50 mM MES at pH 6. Load supernatant containing 5 mg minibody per mL of MMC. Wash with 50 mM MES at pH 6. Elute with a step to 20 mM Tris and 75 mM NaCl at pH 8.5. Clean with 2 M guanidine at pH 5.5. Sanitize with 1 M NaOH. Store in 20% ethanol. Note that the high elution pH has two purposes: to weaken cation-exchange interactions and reduce the salt concentration required for elution, and to condition the sample pH for a subsequent AX step.

Intermediate Purification with UNOsphere Q: Dilute MMC eluate 1:3.5 with 20 mM Tris at pH 8.5. Equilibrate column with 20 mM Tris at pH 8.5. Load sample. Wash with 20 mM Tris at pH 8.5. Elute: a 10-CV linear gradient to 20 mM Tris and 225 mM NaCl at pH 8.5. Clean with 1 M NaCl at pH 8.5. Sanitize with 1 M NaOH. Store in 20% ethanol.

Polishing with HA: To AX eluate, add NaPO4 to a final concentration of 5 mM. Optionally adjust pH to about 7. Equilibrate column with 25 mM NaPO4 at pH 7. Load sample. Wash with 25 mM NaPO4 at pH 7. Reequilibrate with 10 mM NaPO4 at pH 7. Elute with a 10-CV linear gradient to 10 mM NaPO4 and 1 M NaCl at pH 7. Clean with 500 mM NaPO4 and pH 7. Wash with 1 CV water. Sanitize with 1 M NaOH. Store in 20% ethanol and 10 mM NaPO4 at pH 7.

We initially evaluated the AX step in a flow-through format and achieved adequate minibody recovery at pH 7.0 and 12-mS/cm conductivity. These conditions fall within a range demonstrated to support effective reduction of nonenveloped retrovirus (38), but we ultimately chose to run AX as a bind–elute step to eliminate contaminants that bound more weakly than the minibody — to further enhance virus removal and improve reproducibility. This decision was driven mainly by the variability of loading and elution selectivity at the MMC step. But the bind–elute format also contributed to overall process economy by concentrating product and reducing sample volume going into the HA step.

Finally, we returned to the MMC step and evaluated alternative elution procedures. Although we were precluded from exploring conditions that would affect the level of contaminants eluting in advance of the minibody, latitude remained to reduce levels eluting after it and thereby minimize the contaminant load going into the AX step. In short, we discovered that eluting with NaCl significantly reduced dimer content (Figure 7), so we modified the process accordingly. Table 1 summarizes product and contaminant distribution throughout the process. Figure 8 shows PAGE results, Figure 9 shows analytical SEC results, and Table 2 summarizes the recovery.