Standardized Testing of Silica as a Base Material for Difficult Bonded-Phase Preparative Applications

Aug 01, 2014

In high performance liquid chromatography (HPLC) column development, the bonded-phase silica is often only investigated to determine the suitability of a packed column for a particular separation class and not much thought is given to optimization of the base silica to make the overall packing material more suitable for the assay. Here, we examine general-purpose standardized tests to determine the quality of bulk silica before and after bonding. Base silica features such as mechanical strength, acid and alkaline durability, loadability, and overload characteristics are a few of the parameters evaluated that have an impact on the final separation and purification characteristics for a difficult test analyte such as insulin.

Table 1: List of silica particle parameters.
Despite improvements in polymeric particles, the introduction of organic–inorganic hybrids and investigations of other inorganic oxides (for example, titania and zirconia), totally porous and superficially porous silica gels still dominate as the favoured base material in high performance liquid chromatography (HPLC) (1). The properties of silica, outlined in Table 1, make it an ideal packing base material for bonded-phase chromatography, which is by far the most popular approach for analytical and preparative separations. For example, more than 90% of chromatographers use reversed-phase liquid chromatography (LC) in their daily work (1). Even though spherical silica has been around for several decades, manufacturers are still improving silica particles to allow them to be used for even more demanding separations and purifications.

Although the actual number of analytical applications using bonded silica columns exceeds the number of preparative applications, for industrial purifications using porous silica-based packing materials, the amount of silica gel consumed worldwide greatly exceeds the amount of silica used to pack HPLC and ultrahigh-pressure liquid chromatography (UHPLC) analytical columns. In addition, although the properties of the packings used in preparative chromatography share similarities with the optimum properties of analytical chromatography, there are some properties that are more demanding when it comes to large-scale purifications. The purpose of this instalment is to discuss the properties of silica gel that must be improved further and then carefully controlled to meet the demanding applications in large-scale industrial purifications. For our test example, the purification of insulin was chosen since it is the most important large-scale process purification using bonded-phase silica gel chromatography.

The Importance of Insulin Production

Diabetes is an incurable, life-threatening disease. The increase in individuals who are and will be affected by this disease correlates with the worldwide rise in obesity and the growing number of people with access to modern medications. The number of diabetes patients is projected to balloon to a staggering 500 million in the coming years.

The most widely used medication for diabetes is insulin. This peculiar biomolecule comprises 51 amino acids with a molecular weight of a little less than 6 kDa. Insulin is considered either one of the biggest peptides or the smallest of the protein molecules. By its nature, the insulin molecule has a strong tendency to form dimers and further undergo aggregation-coupled misfolding (oligomerization) to form a cross-β assembly. This process is referred to as fibrillation and can complicate the manufacturing process as well as its chromatographic purification.

Insulin manufacturing is a standalone industry these days. Human insulin or its slightly modified variations are cloned into microorganisms. After the expression, insulin is harvested and must be rigorously purified not only from the fermentation broth residues but also from the misshappen isoform impurities. Such difficult separations can be performed using process-scale HPLC with huge dynamic-axial compression columns packed with silica-based reversed-phase stationary phases. Interestingly, the insulin manufacturing industry gobbles up a huge part of the total acetonitrile consumption of the world and almost half of the total spherical reversed-phase silica globally produced.

The biggest technological challenge for insulin manufacturers is the insulin molecules fibrillating on top of the chromatographic column, forming a tough, chewing gum-like layer that blocks the solvent flow and increases the column back pressure. Currently, the solution, though not preferable for silica gel-based packings, is to use a pH 13 sodium hydroxide solution to dissolve and remove this layer blocking the column. This procedure combined with the generally higher than average pressure of the process can damage the silica-based stationary phases badly. Thus, there is a need to develop special stationary phases to address these challenges.