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 RPHPLC 2 at 530 g/h
Minimum yield of 88%
required
Assume 100% binding
4600
4500
Insulin Production (kg/yr)

4400
4300
4200
4100
4000
3900
3800
85
90
95
RP-HPLC 2 Percent Recovery (%)
Fig. 1: Overall annual insulin
production as a function of the RPHPLC 2 unit recovery
100
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).
Design Considerations

Packing (Stationary Phase)

Lipophilically modified silica gels
commonly used



Fig. 3: Structure of C18 resin
Alternative option is Amberchrom HPR10


For insulin purification, C8 to C18
yield best results
Ideal particle size ≤ 12 µm and
pore sizes of 120 to 150 Å
Recommended for polishing stages of insulin purification
Both display similar performance and cost


More data available for silica-based materials
C8 packing selected
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 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
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)
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
Eluting
Loading
Regenerating
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



Average lifetime: 300 cycles


18 kg per column
Quoted price of $8.02/g
7 re-packings required annually
Chemicals

High purity water (WFI): $50,000/year



120 kg WFI/h
$0.05/kg
Note: Neglected acetonitrile, acetone, and acetic acid
Final Design – Cost Summary
Table 1: Summary of costs associated with RP-HPLC 2
Item
Qty
Capital Cost
($)
TechniKrom 35 L column
4
320,000
Kromasil C8 packing
4x7
4,200,000
High purity water
Total cost
Operating
Costs ($/yr)
50,000
320,000
4,250,000
Questions?