In high performance liquid chromatography (HPLC) column development, oftentimes the bonded-phase silica is 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.
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 favored
base material in high performance liquid chromatography (HPLC) (1). The properties of silica, outlined in Table I, 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.
Table I: List of silica particle parameters
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 are similar to 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 installment 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 misshapen 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.