Full Text (PDF)
Most of all, we were gratified that MMC delivered the same purification performance we had observed with CCS but containing 10-fold less product. This greatly lessened our concerns about lot-to-lot feed stream variability rippling through the process. Dimers and aggregates were essentially absent from the final product, as expected. We were initially concerned by an ordered series of lower molecular weight bands on nonreduced PAGE gels, but they were revealed by Western blots to be product-related. This phenomenon also occurs frequently with hIgG1 and hIgG4, and it appears to be a PAGE artifact attributable to disulfide scrambling during sample preparation, even in the absence of reducing agents (39).
Future DirectionsThis study highlights some key challenges with early phase purification process development. Cell lines and culture conditions often are not fully optimized. Product concentrations are frequently low (sometimes very low), and serum supplementation is common with mammalian cells. Despite these burdens, a product's surface chemistry and interactions with various chromatography media can be presumed to be consistent across platforms. Results of elementary scouting experiments can thus provide valuable guidance concerning the fractionation potential of the methods surveyed, reveal relative complementarity among them, and suggest process sequences that preclude extra processing steps such as concentration or diafiltration. These results also provide a preliminary indication of significant ranges for important process variables to support meaningful DoE optimization campaigns. Finally, early CCS in most respects represents the worst-case feed stream a purification process will encounter. Development of an effective process at this stage thereby reasonably ensures later success.
Pending favorable clinical results, we anticipate that cell culture conditions will be modified to eliminate serum supplementation. We suspect that the loading-related variability of MMC binding and elution selectivity is an unfortunate coincidence of this particular minibody sharing similar adsorption/desorption isotherms with BSA. Our interpretation is consistent with the inability of AX and CX to provide substantial fractionation of these solutes. Without BSA, we hope that MMC capacity will roughly double, and reproducibility issues will cease to be a concern. Refinement of elution conditions should then support much higher purity and perhaps further dimer reduction.
We anticipate conserving the AX step by virtue of its regulatory recognition for DNA and virus removal, although we may convert it to flow-through mode on a monolith if the contaminant load following MMC is reduced as expected. We also anticipate conserving the HA step unchanged except for conversion to a step gradient, mainly for its ability to remove aggregates, but also for removing multiple logs of DNA, endotoxins, and viruses. Given the orthogonal mechanistic relationship of HA to AX, their combination promises to be especially effective for virus removal.
Does this procedure offer platform potential? Bacterial, yeast, and mammalian cell cultures each represent different challenges. It is reasonable to expect that chemical differences among minibodies will compound those and other differences. Preliminary results with another minibody, diabody, Fab, and F(ab')2 nevertheless suggest that MMC has broad capture potential for small immunological constructs. HA has meanwhile demonstrated utility for aggregate removal from immunological constructs ranging from minibodies to IgM (8,9) while supporting parallel removal of DNA, endotoxins, and viruses, so it seems reasonable to expect the same with other constructs. The ability of AX to remove DNA and viruses seems likely to translate well across constructs. Taken together, these points suggest that the present procedure may indeed have platform potential.
Results from this study also suggest reassessment of the role of multimodal methods in process chromatography. Mixed modes have frequently solved purification problems that traditional methods could not, but typically not until after extensive time-consuming efforts with traditional methods have proven futile. A growing number of presentations and publications (including this one) suggest that process developers could use their limited time and resources more effectively by including mixed modes at the earliest development stages (8,22,23,25,40). The broad success of HA for aggregate removal recommends initial scouting with NaCl gradients at low phosphate concentrations.
The growing number of capture applications on charged-hydrophobic–hydrogen bonding mixed modes like MMC recommends initial scouting with arginine gradients. Arginine simultaneously affects all three binding mechanisms. If arginine results are promising, then follow-up evaluation can include attempted elution with nonionic eluants such as propylene glycol (which principally affects hydrophobic interactions) or urea (which strongly affects hydrogen bonds too) or pH and salts such as NaCl (which principally affect the electrostatic component of binding) (41). Even if these agents fail to elute the product of interest, they may support washes that significantly improve overall process performance.
These suggestions accurately imply that development of mixed-mode methods is more complex than traditional methods. But they also reveal that mixed modes extend purification capabilities into dimensions beyond the scope of traditional methods. With emerging product classes diversifying rapidly beyond antibodies, these capabilities should receive an enthusiastic welcome.
