This approach imparts greater selectivity than using the eluent volume approach alone, but is still dependent on the reproducibility
of the column's elution profile, because the measurement is not specific for the product of interest. Although the variability
is lower than with the approach of collecting product based on column eluent volume, the lack of a high level of selectivity
still leads to conservative collection setpoints that reduce product yields.
Measuring product purity by on-line HPLC.
The critical quality attribute of the product is its purity. Meeting a specified purity value is the criterion that determines
if the process chromatography step has been successful and if the in-process material is suitable for forward processing.
By transferring the specificity of HPLC to an on-line analyzer, it is possible to directly measure the critical quality attribute
in near–real time, thus allowing the process decision—when to start and stop collection of the product eluting from the process
chromatography column—to be based on the critical quality attribute (product purity) rather than being based on a surrogate
measurement that is affected by the process variability.
Using On-Line HPLC to Automate Process Chromatography Operations
Figure 1 is a diagram of how product purity data generated by the on-line HPLC are transmitted to the distributed control
system (DCS), where the data are compared to a product purity setpoint to determine when to start and stop the collection
of the product based on the critical quality attribute (i.e., product purity). The diagram also shows the typical critical
operating parameters for the unit operation such as conductivity (CT), flow rate (FT), pressure drop (PT), and eluent pH (pH)
that are also transmitted to the DCS where feedback control of these parameters is performed to minimize process variability.
A parameter that is also controlled, but not shown in this diagram, is the eluent feed to the column if the column is eluted
by application of a mobile-phase gradient.
Figure 1. Control of process-scale chromatography column using on-line HPLC to determine product purity
In this operation, the on-line HPLC sends the product purity value, derived from the on-line HPLC data, to the DCS. The product
purity value generated by the on-line HPLC is compared to the product purity setpoint in the DCS. If the product purity is
lower than the product purity setpoint, the DCS sends a signal to the three-way valve in the column eluent stream to divert
the eluent stream to waste. If the product purity is greater than the product purity setpoint, the DCS sends a signal to the
three-way valve in the column eluent stream to divert the eluent stream to the product collection tank. By basing the mainstream
pooling decision on a direct measurement of product purity, variability in the mainstream pool purity is minimized. Thus,
by varying the timing of output collection according to a near–real time measurement of product purity determined by the on-line
HPLC analyzer, the process can accommodate variability in the process input and process operations. The dependency of product
purity on the operation of the process chromatography column is significantly reduced when compared to collecting the product
pool based on elution volume or a combination of elution volume and an A280 value.
By using the on-line liquid chromatography approach to provide near–real time information on product purity, a more efficient
and robust process is achieved which provides more consistent product purity. This is accomplished in several ways:
- Eliminating fraction collection significantly reduces opportunities for errors and contamination
- Eliminating off-line analysis of the fractions reduces cycle time
- Yield may be increased by having an on-line, mainstream collection measurement that is more specific to the product, because
this allows a larger amount of the elution peak to be collected when compared to measurements such as elution volume or A280
that are not product-specific.
- The process endpoint—determined by achieving a particular product purity value—is flexible and can adjust to variability in
the process input and operations.
- Increased levels of process automation (e.g., automated sequencing) can be implemented, thus leading to more precise operations
than typically can be achieved with manual operations.