Quality by Design in the CMO Environment - - BioPharm International


Quality by Design in the CMO Environment

BioPharm International
Volume 20, Issue 12

The study results showed the Octyl chromatography to be quite robust. It was insensitive to pH from 6.0 to 7.0, linear flow rate from 100 to 180 cm/hr, and bed height from 15 to 25 cm. The bed height and flow rate combine for a range of residence time from 5 to 15 minutes with no impact.

Table 5
Increasing loading density, as expected, reduced the resolution of species A from product. Less resolution, in turn, resulted in fewer fractions pooled and thus lower yield. There was a strong correlation between high loading density and early elution. Low ammonium sulfate in the load was correlated with early elution and improved resolution of product from species A, possibly because the product tended to elute early. In contrast, species A eluted at about the same point in the gradient regardless of loading conditions. The trends discussed above, which are highlighted in yellow in Table 5, were significant whether or not the percent species A in the load was included in the analysis.

Additional trends were revealed with the inclusion of percent A in the load. Higher temperatures favored better yield, presumably because of the general phenomenon observed with HIC resins of improved binding of protein at higher temperatures. There was also a correlation between higher temperature and later elution, probably caused by the same mechanism. Larger amounts of species A in the load led to lower yield because fewer fractions could be pooled. The correlations of species A with early elution and less resolution of species C do not have readily apparent explanations. They may be collinear with other factors. No two-factor interactions were revealed in the analysis of the data. The targets for the key variables determined for this study were loading density 10 g/L of resin, 1.1 M ammonium sulfate in the load, and temperature ≥18 C.

Figure 4
None of the variables tested had any impact on resolution between product and species B. Octyl Sepharose Fast Flow may be incapable of better separation, or the controlling parameter has yet to be discovered. Resolution of species C only seemed correlated with the percent A in the load. These observations reaffirm that the primary role of the Octyl column is to remove species A; other species may be reduced, but not removed, except at a high cost to yield. However, as long as the key variables are controlled, the prediction profiler generated by JMP 6 calculates that the resolution of species A should vary only between 0.53 and 0.59 for 6.9–12.0% A in the load for the Octyl column.


Table 6
Following development of the purification process, four manufacturing runs were conducted at the 2,000-L culture scale. The bed volume of the Octyl column was 40.5 L. Figure 4 is an overlay of the UV absorbance chromatograms of the Octyl step at manufacturing scale, and Table 6 shows the major input and output variables for the runs. Column dimensions and temperature were constant, and flow rate was <150 cm/hr to maintain pressure less that 2 bar.

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