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.
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.
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.
ANALYSIS OF MANUFACTURING RUNS
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.