Kromasil^® notes

This technical note presents the purification of Tirzepatide using Kromasil^® 100‑13‑diC4, a reversed‑phase stationary phase designed to deliver robust performance for large-scale, hydrophobic peptide separations.

The study outlines crude material preparation, analytical characterization, preparative loading strategy, and fraction evaluation. The resulting data demonstrate the capability of Kromasil^® diC4 to deliver high purities and tunable recovery, offering a practical and scalable solution for peptide purification processes.

To test the durability of Kromasil^® 100 Å C4 material, it was packed and re-packed multiple times into a 50 x 250 mm DAC column. When silica is repeatedly exposed to high pressure and shear forces during slurry preparation and packing there is risk of crushing the material. This is especially the case for low quality silica where the mechanical stability is poor. Kromasil however can be packed and re-packed many times without breaking the silica to small fragment and thereby avoiding an increased pressure over the column.

When dealing with UHPLC/ HPLC analysis of drug samples, selectivity and resolution are key parameters. Reversed phase analysis based on silica stationary phases is the most common technique but it is limited to acidic and neutral pH conditions. Kromasil^® EternityXT UHPLC/HPLC columns can operate within a wider pH window; therefore, we illustrate the benefit of using these columns to exploit selectivity and resolution at high pH to analyse a mixture of alkaloids.

An example on how the Kromasil® GLP-1 agonist purification toolbox can be used to boost the purification of liraglutide, a common GLP-1 receptor agonist in pharmaceutical applications.

A C18 might be the most versatile phase and a first pick when developing analysis or purification methods for HPLC. However, when working with biomolecules and polar compounds, one may need to play with other parameters than hydrophobicity, like total charge at various pH as well as being able to run in highly aqueous conditions. In those situations, the boundaries of a traditional silica-based C18 might feel slightly too limiting. Kromasil offers therefore alternative C18 options to cover extended conditions: Kromasil EternityXT C18 and Kromasil Classic C18(w).

List of applications using Kromasil phases for the separation of oligonucleotide:

Silica based chromatography materials are typically considered stable in the range of pH 2 to pH 8 and separations and purifications are usually carried out under these conditions. However, there are applications where higher pH could be used to achieve better purifications to attain higher purity and/or yield. EternityXT stationary phase materials are ideal for production facilities that need to work with an extended range of pH because it is stable under low and high pH conditions.

UCB is a global biopharmaceutical company dedicated to the research, development and commercialization of innovative medicines with a focus on the fields of central nervous system disorders and immunology disorders.
At UCB Biopharma in Braine-l’Alleud (Belgium), one of the two research centers, day after day, numerous intermediates and potential Active Pharmaceutical Ingredient (API) candidates are sent to the Preparative Chromatography Team for purification.

The main proportion of all synthetic pharmaceutical APIs are basic in nature, and will in many cases exhibit an increased loadability, and hence productivity, at high pH. Basic peptides, oligos, PNAs, etc, will also benefit from high pH separation methods.

Traditional preparative liquid chromatography was born in 1903 when the Russian botanist M. Twsett made the first separation of carotene pigments. The columns he used were made of glass, they were packed with gravels and were basically operated at atmospheric pressure. This situation kept unchanged for many years, until the 1960’s when organic chemists began to use stainless steel columns operated at a few bars.

A nice scale-up example from column i.d. 4.6 to 21.2 mm.

In the last issue of KromaNews we looked at the importance of performing overloading studies during preparative HPLC method development. It was shown that switching the stationary phase, while keeping all other chromatographic parameters constant, altered the non-linear adsorption behavior.
     In this issue, the adsorption behavior of a peptide on the stationary phase at different buffer pH will be shown.

In order to have a seamless and problem-free scale-up, and to be able to establish an efficient and cost-effective production, one must assure that the large scale column used is packed with the same high efficiency as the column used for the method development. And this can relatively easy be done with today´s DAC columns available on the market!

Preparative HPLC differs from analytical HPLC since it needs to be performed in the nonlinear part of the adsorption isotherm in order to maximize important parameters such as column loading and productivity. In preparative HPLC, method development screening is commonly performed with analytical injections. This makes it possible to establish and compare selectivity in different chromatographic systems. From the screening, one or more candidate phases are selected for a closer preparative study with overloaded injections. This case study illustrates the risk of relying only on selectivity during preparative method development.
The scope of the study was to separate an API (Mw ≈ 900 g/mol) from its impurities and achieve 99.0% purity with as high productivity as possible.

Obesity has developed into a severe, global health problem during the last decades, and as a result pharmaceutical companies have tried to develop a remedy for obesity, that can be used in combination with dieting and physical exercise. The one medication currently available and approved for long-term use is an anti-obesity drug marketed as Xenical or Alli. The API in the drug is Orlistat, also known as tetrahydrolipstatin.

In recent years, India has proven to be an area of strong growth in the peptide market. Numerous Indian companies are developing biosimilars while several others are focusing on research and development (R&D) of novel therapeutic peptides. Issar Pharmaceuticals, located in the Hyderabad, India is an example of a company that has specialized in providing novel therapeutic peptides. The company has successfully synthesized a number of promising peptides in its pipeline, most notably being the Melgain^®, which was commercialized in 2004.

One of the beauties of purification using preparative HPLC is the simplicity of how scaling of a separation process is made. This article will give you an insight into how this is done, and which difficulties may occur.

Kromasil packing media are today the dominating materials used for large-scale production of human insulin and variants, among many other applications. Multi-ton volumes of Kromasil per year are used globally to produce these APIs, with high purity demands.
This article will show an example of the development work that has been performed to facilitate cost-effective processes for the final purification of human insulin, and to service our customers in order to lower their costs.

With over 25 years of expertise in the development of preparative chromatography equipment and purification processes, Novasep has pioneered the field of industrial HPLC including both batch and continuous (SMB) technologies. Within recently extended facilities in Shanghai, the company offers process development and scale-up services for the production of APIs under cGMP conditions.

In order to develop a successful preparative HPLC method one first needs to define relevant goals for the specific purification. The most important and common parameters defining the quality of a separation is loading (defined as m_crude/m_resin), purity and yield. Purity is usually already defined by regulatory agencies (such as FDA or equivalent) and often includes area percentage of product as well as individual impurities, while yield simply is the ratio of purified product divided by amount loaded onto the column.

In preparative chromatography it is of uttermost importance that all parameters are optimized to give the lowest possible cost for the target substance purified. We have in previous articles described the importance of optimization of the HPLC separation. However, another factor that could greatly influence your separation cost is the lifetime of the packing material. To run the separation using a stable packing material will not only result in a lower cost due to a longer lifetime of the packing material, but the separation can also be run with less disturbances, with more stable conditions for a long time.

Major changes are occurring in the development of new therapeutics within large pharmaceutical and biotechnology companies. These companies are becoming leaner and rely more on outsourcing, anything from analytical methods to full-scale contract production. With the change in paradigm there is a need for contract manufacturers (CMOs) to expand their services and guarantee quality work. Experience allows for contract manufacturing companies to deliver anything from robust analytical methods to purified APIs at a reasonable cost.

A common question that often arises in the discussion between prep chromatographers is when and why one should choose either isocratic or gradient elution? Since we in the Kromasil group have a long experience of developing large scale separations we would like to share some of these experiences with the readers of KromaNews.

Quite often one meets people with excellent knowledge and experience in analytical HPLC, and while much theory and underlying equations can be used on a preparative scale, a lot may differ. The main difference is the scope of the separation. In analytical chromatography you are most certainly looking for information in some way, either identification or quantification (or both) of one or several peaks in one chromatogram. However, in preparative chromatography the isolation of a single substance at a certain purity is usually the main goal. This difference has a large impact on how to develop the chromatographic method in order to optimize factors such as yield, purity and loadability.

During the last one and a half years Separation Products has undergone some important changes, as Domingo Sanchez, one of the founders of the group, has retired as General Manager.
Domingo is now replaced by Sylvia Winkel Pettersson, who has been working in the Kromasil group since 1999 in different positions. Sylvia hence has a strong background and experiences to support her as Separation Products' new General Manager.

A chromatographic system is defined by the nature of the stationary and mobile phase. While there are a large, although discrete number of stationary phases to choose from, the selection of the right mobile phase composition renders almost infinite possibilities. We are elucidating one of the important parameters, namely the buffer capacity of a reversed phase eluent.

The recent years has seen a tremendous increase in the use of Industrial Scale HPLC, especially in emerging countries like India and China. Many large DAC columns have been installed, and are run using short bed lengths, together with small, spherical, silica-based particles, and relatively high flow rates. This is what we call the High Performance Concept in preparative HPLC.

Separation Products has had a long collaboration with the group of Professor Cesar Santana at the Universidade Estadual de Campinas in Brazil. Professor Santana and his group have produced several scientific papers in the field of large scale purification of enantiomers. We are now proud to give the readers of KromaNews a flavor of the work of Professor Santana's group and some nice pictures from the lovely group in Brazil.

Kromasil offers the lowest possible total separation cost by providing the best silica-based HPLC, SMB and SFC packing material. Superior loadability and lifetime let you boost productivity and reduce costs.

Mobile phase additives are commonly used in analytical chiral separations without trouble. But in preparative scale, additives make solvent recycling tricky and can cause re-racemization. By using Kromasil AmyCoat or CelluCoat it is possible to exclude additives in chiral preparative separations.

When Hybio was founded in 1998, it was one of the first companies in China to enter the peptide industry. Today, Hybio is a full-scale peptide producer that invests heavily in research and development. Kromasil is part of their success by providing silica for efficient and reliable purifications.