Biomedical Engineering Reference
In-Depth Information
the chromatography column packing integrity as explained in more detail in the
following section.
12.8 “RELEVANT TIME” COLUMN INTEGRITY MONITORING
(MOMENTS ANALYSIS VERSUS HETP)
Monitoring the quality of packing and detecting integrity breaches in purification liquid
chromatography columns is important from process performance and final product
quality perspective. Conventional techniques for this task include monitoring a few key
variables such as step yields, prepool volumes, and pulsed-injection height equivalent of
a theoretical plate (HETP) testing [68, 69]; or simply inspecting the chromatograms
visually. Transition analysis uses existing process data (solute signals such as pH,
conductivity, and UV) during buffer transitions at the column outlet during a chroma-
tography operation to assess the packed bed integrity. The postcolumn signal (e.g.,
conductivity) is noise filtered and normalized, and its derivative is taken to calculate
several parameters that are indicative of the packed bed integrity. These calculated
parameters are orthogonal and include the following:
1. Number of inflection points, N
2. Maximum rate of change or maximum derivative (dC/dV)
max
3. V
max
4. Breakthrough volume (BTV)
5. Cumulative error or peclect number
6. Non-Gaussian HETP
7. Gaussian HETP
8. Asymmetry factor
9. Skewness
10. Kurtosis
11. Overall integrity
These transitional measures are much more sensitive than pulse injection-based
HETP and asymmetry results enabling an early detection of column integrity deteriora-
tion during its use at each cycle. Depending on the chromatography column type, there
are multiple opportunities to perform transition analysis within the process phases
including equilibration, elution, and the clean to store step.
12.8.1 Number of Inflection Points
The number of inflection points during a transition is determined by counting the number
of peaks from a plot of (dC/dV) versus V. All those peaks higher than 10% of (dC/dV)
max
are counted. An increase in the number of inflection points indicates early breakthrough
that is associated with an integrity breach.
