Reversed Phase HPLC 2

1
Reversed Phase HPLC 2

• CHEE 450 Engineering Biology
• Thomas Cooper
• Pedro Isaza

2
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

3
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

4
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

Fig. 1 Overall annual insulin production as a
function of the RP-HPLC 2 unit recovery
5
Design Considerations

• General Operating Parameters
• Temperature range of 15 to 20 oC
• Pressure range of 20 to 100 bar

• Column Type
• Fixed-bed vs. Axial Compression
• Large size creates difficulties for efficient and
  reproducible packing
• Axial compression eliminates this problem

Fig. 2 Illustration of self-packing axial
compression column (TechniKrom, 2007).
6
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 Å

Fig. 3 Structure of C18 resin

• Alternative option is Amberchrom HPR10
• Recommended for polishing stages of insulin
  purification
• Both display similar performance and cost
• More data available for silica-based materials
• C8 packing selected

7
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

8
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 insulin solubility
• Loading, Elution, and Regeneration Scheme
• Load insulin onto column in aqueous-alcoholic, or
  purely aqueous, buffer solution
• Linear gradient ranging from 15 to 30
  acetonitrile effectively separates insulin in 1
  CV or less
• Regeneration with 60 acetonitrile and pH 7.4

9
Design Considerations

• Final Yields
• Minimum 88 yield required
• Yields from 83 to 98 documented for C8

• Confirmation
• Product stream monitored via spectroscopy
• At 280 nm for insulin

Insulin Peak
Fig. 4 Elution of insulin and insulin
derivatives under acidic conditions (Kroeff et
al., 1989)
10
Final Design Batch Time

• To find number of columns required for continuous
  operation
• At inlet flowrate loading time is 1 h
• Elution in 1 CV at 1.5 CV/h elution time is 40
  min
• Similarly, column regeneration time is 40 min
• Total HPLC cycle is 140 min

• 3 columns are required
• 4 recommended for unexpected failures

11
Final Design Cost Analysis

• Axial compression column
• 32 L required
• TechniKrom 35 L (30 cm ID x 50 cm) 80,000
• Packing
• Kromasil C8 (10 µm, 100 Å) 150,000/column
• 18 kg per column
• Quoted price of 8.02/g
• Average lifetime 300 cycles
• 7 re-packings required annually
• Chemicals
• High purity water (WFI) 50,000/year
• 120 kg WFI/h
• 0.05/kg
• Note Neglected acetonitrile, acetone, and acetic
  acid

12
Final Design Cost Summary
Table 1 Summary of costs associated with
RP-HPLC 2
13
Questions?