ABSTRACT

This article discusses how on-line high-performance liquid chromatography (HPLC) can measure product purity in the column eluent stream in near–real time. These data can then enable the automation and control of a purification column operation, thus reducing product variability, shortening process cycle time, and increasing yield. An example application demonstrates how on-line HPLC is used as a process analytical technology to ensure the process can accommodate variability in the separation while ensuring the product meets its critical quality attributes.

Pharmaceutical products, particularly those derived from biotechnology based processes, frequently use process chromatography in downstream operations to isolate and purify the product of interest. The high selectivity provided by chromatography is especially important when dealing with the complex streams encountered in biotech processes in which the product of interest often is a minor process component at the initiation of downstream isolation and purification operations. Knowing when the product of interest at the targeted purity elutes from a process-scale purification column can be difficult to determine because these separations rarely provide baseline separation of the product of interest from other components in the process stream.

Determining when product elution occurs generally involves collecting the column eluent stream into small-volume time slices or "fractions" that are analyzed off-line in the laboratory by means of higher resolution and frequently orthogonal chromatographic techniques such as reversed phase chromatography. This process of eluent fractionation and off-line chromatographic analysis is performed during process development to develop sufficient process understanding so that developers can predict when the product will elute from the process scale chromatography column. The eluent fractionation and off-line analysis process often continues even after the product moves into manufacturing.

Fractionating the column eluent has several disadvantages, including:

• Labor intensive
• Opportunities for errors to occur
• Increased risk for product contamination and exposure of operators and facilities to process components
• Increased process cycle time caused by time needed to obtain off-line assays
• Suitable storage required for "work in process" (the column fractions)
• Degradation of product caused by these delays
• Reduced production capacity resulting from long cycle times
• Difficult to automate processing because of manual handling of fractions.

Because of the many negative issues associated with column fractionation, companies strive to eliminate this operation. Options to eliminate fractionation include collecting product based on column eluent volume; collecting product based on column eluent volume and optical density; and measuring product purity by on-line HPLC.

Collecting product based on column eluent volume.

In this approach, the eluent volume where the product of sufficient purity is expected to elute is determined from historical data; the start-collection and stop-collection setpoints are set based on these empirical data. An in-line flow meter measures the eluent volume to control the collection of the product of interest as it elutes from the process chromatography column.

The difficulty with this approach is that it is totally dependent on the reproducibility of the column elution profile which is affected by many variables. These variables include column loading, the purity of the starting material, the affinity of various components in the process stream for the stationary phase, and the column's operating parameters such as the generation of the gradient used to elute the column. Because of variability in these inputs, it can be challenging to maintain reproducible purity levels of the recovered product when using this approach, even with feedback control of the column's critical operating parameters (including gradient generation). This variability generally leads to conservative collection setpoints that reduce product yield.

Collecting product based on column eluent volume and optical density.

In this approach, an in-line ultraviolet (UV) sensor set to monitor absorbance at 280 nm measures the amount of product present in the eluent stream by relating the absorbance at 280 nm (A280) to product concentration. Because this measurement is not product-specific (i.e., any peptide in the stream will generate an A280 absorbance signal) the output of the optical density (OD) sensor output is examined in combination with the in-line flow meter to improve resolution. That is, the product is only collected when the OD at 280 nm exceeds the OD setpoint and falls within the eluent volume setpoint window.