Routine Implementation of Very-High-Pressure Liquid Chromatography in Pharmaceutical Development

High-pressure liquid chromatography is probably the most widely used analytical technique for characterizing drug substances and drug products in the pharmaceutical industry. It is widely employed across all stages of drug development, from drug discovery to pharmaceutical development and eventually commercial manufacture. Though applications may vary between discovery and development, there is a consistent need to increase the productivity and speed of HPLC analyses. One of the most efficient approaches to speed up HPLC analysis without sacrificing efficiency is to decrease the particle size.1 Currently, LC columns packed with 3–5 µm particles remain the workhorse analytical columns. In the late 1990s, Jorgenson’s group first demonstrated that much higher-efficiency separations could be achieved using capillary columns packed with sub-2-µm particles.2,3 Subsequently, Lee’s group demonstrated that such columns could also be used to provide very fast separations.4,5 The main drawback is that the required backpressure to run such columns is well above the limit of conventional HPLC instruments, which is usually around 5000–6000 psi.

In recent years, most chromatographic instrument vendors have been offering commercially available LC instruments that provide backpressures ranging from 9000 to 17,000 psi. Meanwhile, LC columns packed with sub-2-µm particles became commercially available with a much wider range of column chemistries and dimensions. A variety of terminology has been used by vendors and in the literature. For the purposes of this article, the generic term “very-high-pressure liquid chromatography” (VHPLC) will be used to reference these newly developed LC instruments that can provide backpressures above 6000 psi.

VHPLC is a natural extension of HPLC and is governed by the same chromatographic principles. With more VHPLC instruments and columns becoming available, the technique has gained popularity in the pharmaceutical industry.6–9 In pharmaceutical development, analytical methods are required to be validated according to regulatory guidelines, and are often transferred to worldwide manufacturing sites and contract vendors. These requirements provide some unique challenges in implementing VHPLC technology in pharmaceutical development. The following examples illustrate how to address such challenges during VHPLC method development, validation, and transfer.

VHPLC method development

A common HPLC application in pharmaceutical development is impurity profiling of drug substances and drug products, which requires that all critical impurities be separated from the active pharmaceutical ingredient (API) and from each other. Since some impurities may have structures very similar to the API, such as diastereomers and regio-isomers, it can be difficult to achieve satisfactory separation, and typical separation times can range from 30 to 90 min. For these separations, VHPLC has been investigated to shorten the run times to provide increased productivity. Accordingly, the method of impurity profiling will be used here to illustrate one VHPLC method development strategy.

To smooth the transition from HPLC to VHPLC, a VHPLC method development strategy was needed that was able to retain most of the choices in HPLC method development. The parameters affecting resolution in reversed-phase LC include column efficiency, column chemistry, organic solvent, mobile phase pH and additives, column temperature, solvent strength for isocratic separation, and gradient profiles for gradient separation. Since column efficiency is roughly proportional to the ratio of column length to particle size, and the process typically starts with 150-mm-long columns packed with 3- or 3.5-µm particles for HPLC impurity method development, a 100-mm-long column packed with sub-2-µm particles is a good choice to begin on the VHPLC side. Compared with HPLC, one limitation to VHPLC method development is column availability, since some widely used HPLC columns are currently not available in the sub-2-µm particle range, but only in the sub-3-µm particle range with maximum backpressure of around 9000 psi. Because column chemistry is a very important factor for selectivity, column selection was extended to include sub-3-µm particle columns.

Another limitation of VHPLC is the instrument maximum pressure. An HPLC instrument with a 6000-psi pressure limit can easily handle a full-range methanol gradient at room temperature for 150-mm-long columns packed with 3- or 3.5-µm particles; this is not the case for some commercially available VHPLC instruments when 100-mm-long columns packed with sub-2-µm particles are used. To accommodate this limitation, we can decrease the flow rate, increase the column temperature, or limit the choice or ratio of organic solvent. Since organic solvent is an important factor for selectivity, and flow rate directly affects separation time, the best option is to slightly increase the column temperature. Other parameters affecting resolution, such as pH, mobile phase additives, and gradient profiles, are typically not affected by the instruments.

Based on the above, the following VHPLC method development strategy was devised:

Figure 1 - VHPLC impurity profiling method. Insert is an expanded section around API peak.

  • Maintain column selectivity by including sub-3-µm particle size columns for initial screening, and start with 100-mm-long columns
  • If necessary, limit backpressure to under 9000 psi by using a column temperature of 35 °C or higher for initial screening
  • Start with mobile phase pH/additive screening followed by a combined column/solvent screening to optimize selectivity
  • Reexamine whether a shorter column or higher pressure can be applied with the best LC conditions from screening results obtained from the last step
  • Optimize gradient/column temperature with the aid of software such as DryLab (Molnár Institute, Berlin, Germany).

Figure 1 is an example of a VHPLC method developed based on the above strategy. The method provides adequate resolution for critical impurity pairs with a run time of around 10 min. Using this strategy, VHPLC impurity profiling methods can typically be developed within one week.