Reverse-phase high performance liquid chromatography (RP-HPLC) is a technique used for isolation of biologics based on hydrophobicity, and the molecules separate from the mobile phase to the stationary phase containing the hydrophobic immobilized ligands. RP-HPLC is probably the most used analytical method for separation and determination of peptides and proteins in a wide range of applications.
The stationary phase of RP-HPLC mostly comprises non-polar alkyl hydrocarbons such as C4, C8 or C18 chains bound to silica or other inert support. C18 and C8 refer to the alkyl chain lengths of the bonded phase, both of which are the most common hydrophobic bonded phases. The length of the chain affects the hydrophobicity of the sorbent phase, thereby increasing the retention of ligands. C18 has the highest hydrophobicity, and C18 columns are the most used reverse-phase columns in most HPLC labs. Because its carbon chain length is longer, C18 is more hydrophobic. The higher hydrophobicity means a longer retention time of non-polar compounds, and therefore non-polar compounds travel through the column more quickly with C18 than with C8. When shorter retention times are required, C8 is used. The mobile phase is polar, the elution order is polar, followed by less polar and weakly polar compounds, and finally non-polar compounds.
Some RP-HPLC processes require high pH conditions, such as column regeneration solutions containing 0.1M NaOH, which results in a short life of conventional silica media. Hybrid silica particles with a size of less than 10 µm have high pH stability, making them useful for analytical HPLC applications. Few commercial hybrid silica particles in 10 µm or larger sizes are available for process-scale chromatography, probably due to the small specific surface area of such hybrid particles, high manufacturing costs, and technical difficulties in large-scale production. Although hybrid coated silica particles have a longer lifetime than silica particles at high pH, but are still less stable than the hybrid particles.
Amerigo Scientific offers UniHybrid® Eterne monodisperse hybrid silica particles in 10 µm size for the purification of peptides and proteins such as insulins, GLP-1, and their mimics. Based on the innovative Precisely Controlled Silica (PCS) technology, large quantities of UniHybrid®Eterne monodisperse hybrid silica particle can be produced at low cost. UniHybrid®Eterne particles have a high surface area, good mechanical strength, and chemical stability more than 10 times that of conventional silica particles, which can meet the high requirements of RP-HPLC media with high performance and pH tolerance.
|UniHybrid® Eterne 10-120 C18
|30g; 50g; 100g; 300g; 500g; 1kg; 5kg; 10kg; 50kg; 100kg
|UniHybrid® Eterne 10-120 C8
|UniHybrid® Eterne 10-120 C4
Fig. 1. UniHybrid ® Eterne SEM
|C18 / C8 / C4
Semaglutide belongs to a class of drugs called glucagon-like peptide-1 (GLP-1) receptor agonists for type 2 diabetes. It mimics the GLP-1 hormone that is released from the gastrointestinal tract in response to eating. GLP-1 is an incretin hormone that is expressed in enteroendocrine L-cells through post-translational processing of the proglucagon gene product by a prohormone convertase (PC)1/3. GLP-1 is well known for its prominent activity in glucose-dependent insulin secretion in the pancreatic β-cells. Other important biological functions of GLP-1 hormone include induction of proinsulin gene expression, inhibition of glucagon release, stimulation of pancreatic β-cells proliferation, induction of neogenesis, inhibition of β cell apoptosis, and significant delay of gastric emptying.
Insulin is a protein administered subcutaneously to humans for the treatment of type 1 diabetes to control glucose homeostasis, when the production of pancreatic β-cells is insufficient to ensure daily requirements for this hormone. Insulin is a well-characterized protein with a molecular weight of 5.8 kDa and consists of two peptide chains, one chain containing 21 amino acid residues and the other chain containing 30 amino acid residues.
As shown in the figure, compared to another brand of silica particles, UniHybrid® Eterne 10-120 C18 and UniHybrid® Eterne 10-120 C8 showed similar peak width, but better selectivity and resolution for front and back impurities for insulin purification.
Fig. 2. Comparison of UniHybrid ® Eterne 10-120 Cl8/C8 and other silica C18
Column: 4.6 mm x 250 mm. Mobile phase: (A) buffer solution, (B) CAN. Flow: 0.42 ml/min. Sample loading: 14 mg/ml-CV.
Using UniHybrid®Eterne silica particles, semaglutide can be purified through a two-step purification process. In the first purification, semaglutide is purified using UniHybrid® Eterne C8 under basic conditions. The first purification of the sample usually contains more impurities and requires more than 0.1M NaOH wash/regeneration solution. In the purification, UniHybrid® Eterne C8 provides a high yield and a long lifetime.
In the second purification, semaglutide is purified using UniSil® Revo C8 under acidic pH 3.5 condition. The second purification of the sample requires less than 0.1M NaOH regeneration solution. In this step, UniSil® Revo C8 is used to obtain a higher purity of semaglutide.
Fig. 3. Fully synthetic semaglutide purification flow chart
UniHybrid® Eterne 10-120 C18 exhibits excellent high pH stability. As shown in the figure, naphthalene retention time only decreased by ~8% and efficiency unchanged after pH 13 flash for 73 hours. UniHybrid® Eterne C18 maintained good peak shape, efficiency and retention after 73 hours of pH 13 flash while another brand of silica particles based C18 degraded badly after 14 hours of pH 13 flash.
Fig.4. Retention and efficiency changes of UniHybrid ® Eterne 10-120 C18 in pH 13 (0.1M NaOH) flash condition
Fig.5. Chromatograms of other competitor's silica based C18 and UniHybrid ® Eterne C18 in pH 13 flash condition
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