Quality by Design in the CMO Environment - - BioPharm International


Quality by Design in the CMO Environment

BioPharm International
Volume 20, Issue 12

The SEC-HPLC assay was used to determine the percent of product and species A, B, and C present in a sample. A Tosoh G3000SWXL column (catalog number 08541) with a guard column was used to analyze Octyl fractions. The mobile phase used was 100 mM sodium phosphate, 200 mM sodium chloride, pH 6.5. The target load was 30 μg of protein per injection, and total run time was 35 minutes. A gel filtration standard was used (Bio-Rad, 151–1901) to verify system suitability.


Analysis of DOE Runs

Table 3
For each run, SEC-HPLC was performed on the load, unbound, gradient elution, and strip fractions to give a purity profile of each fraction. Additionally, the concentration of each of these fractions was determined by absorbance at 280 nm. Theoretical pools of the gradient elution fractions were then calculated using this data. The first criterion for pooling was that the pool must contain less than 2% of species A, because downstream processes did not demonstrate removal of this impurity. Furthermore, the pool must contain 10% species B and species C combined. In some runs, a pool could not be made to satisfy these requirements, so the yield for those runs was 0%. The yield calculated for the theoretical pools is product-specific, i.e., product in pool product in load x 100. Table 3 shows the raw data from DOE run 2.

Table 4
An absorbance threshold was used as an indicator of where in the salt gradient protein elution occurs, and was expressed as column volumes. The start of protein elution was defined as the point where absorbance at 280 nm increased above 50 mAU. A value of 1 would indicate that the 50 mAU threshold was surpassed at 1 CV into the gradient. A negative value indicates that elution began before the start of the gradient, that is, during post-load wash. The eluate was not collected until the gradient phase of the method, so any protein eluted during the wash was lost, reducing the yield.

Figure 3
To determine resolution between two peaks, the number of CVs between the two peaks was divided by the average width in CV of the peaks being compared. A resolution value of 0 indicates no separation, and a value of 1 indicates complete separation between the species. JMP 6 statistical software was used to analyze the data from the DOE using seven input variables (the original six in the design plus percent species A in the load) and the output variables of yield, elution start, and resolution. Effect screening was used to identify the main effects with a 95% confidence level. Table 4 contains the output variables for each run in the DOE. Figure 3 is the prediction profiler generated by JMP 6 software, which shows trends as parameters were varied. Table 5 is a summary of the analysis with JMP 6.

The percent species A in the load was not included in the original design. However, it was identified as a variable after analysis of fractions by SEC-HPLC. Upstream development was concurrent with downstream development to save time so there was some variability in the feed stream. This turned out to be a boon: adding the parameter of percent A in the load to the analysis revealed additional correlations. Their validity was supported by the large increase in the adjusted R2 value for the models for percent yield (0.30 to 0.74), resolution of species C (–0.35 to 0.65), and start of elution (0.62 to 0.89).

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