Reversed Phase HPLC 2 - Chemical Engineering反相高效液相色谱法2 -化学工程.pptVIP

Reversed Phase HPLC 2 CHEE 450 Engineering Biology Thomas Cooper Pedro Isaza Purpose Alternatives Final insulin product must be purified Mixture contains many impurities Unreacted insulin esters Precursors Deamidated insulin RP-HPLC demonstrated to separate insulin and insulin-like compounds differing by one amino acid Also possible with ion exchange chromatography Loss of product Lower yields How it Works HPLC able to separate based on charge, size, and hydrophobic character Hydrophobic analytes interact strongly with resin Hydrophilic pass freely through the column Organic solvents are used to elute hydrophobic molecules Design Requirements Annual insulin production of 4000 kg at 99% purity Insulin enters RP-HPLC 2 at 530 g/h Minimum yield of 88% required Assume 100% binding Design Considerations Column Type Fixed-bed vs. Axial Compression Large size creates difficulties for efficient and reproducible packing Axial compression eliminates this problem Design Considerations Packing (Stationary Phase) Lipophilically modified silica gels commonly used For insulin purification, C8 to C18 yield best results Ideal particle size ≤ 12 μm and pore sizes of 120 to 150 ? Design Considerations Column Size C8 suggested loading is 17 mg insulin/mL For 530 g/batch, 32 L of packing is required Mobile-phase pH Acidic conditions elute insulin before impurities Early elution improves yields Ideal pH range of 3 to 4 Well below isoelectric point of insulin (pH 5.4) Compatible with chosen silica resin (pH 2 to 8) high pH: dissolution of the silica very low pH: hydrolysis of attached C8 chains May lead to insulin deamidation Not significant problem due to short exposure times Design Considerations Organic Modifier for Elution Requirements: good insulin selectivity and low viscosity Acetone or acetonitrile are recommended Acetonitrile Well-documented analytical insulin separation High yields obtained at production scale Acetone Lower yields due to poor insuli