PRODUCTION OF HUMAN INSULIN:

CRYSTALLIZATIONBy: James Gillis, Keni Mallinen, Brenna Swift

CHEE 450 Design Project

PRESENTATION OUTLINE• Process Overview

• Insulin Crystallization

• Batch Crystallization Equipment

• Design Parameters

• Production Capacity

• Materials of Construction

• Suppliers and Costing

• Sterilization and Recovery

• Comparison to Alternative Unit Operations

INSULIN CRYSTALLIZATION Majority of pharmaceutical products marketed in crystalline forms

• high purity• facilitates subsequent centrifugation and

drying • improves product’s aesthetics

Insulin separation by precipitation using zinc chloride

Rhombohedral, zinc hexamer crystal structure

Batch crystallizers

Similar to stirred tank reactors

BATCH CRYSTALLIZATION EQUIPMENT

OPERATING PARAMETERS: SUPERPRO Conditions, reagents, and their interaction dictate crystallization

rate, yield, size, morphology, etc.

SuperPro parameters compared to conditions and reagents utilized in previous insulin crystallization procedures

No measure of pH in SuperPro; extent of crystallization set manually at 90%

Component Flow rate (kg/batch)

Mass Comp. (%)

Concentration (g/L)

Stream S-168: Stream from Diafiltration Unit OperationAcetic Acid 2.21909 1.2602 12.653585

Insulin 12.78009 7.2579 72.874061

WFI 161.08573 91.4818 918.535910Stream S-126: Charge Stream for Crystallization Tank

Ammonium Acetate

1.13267 36.1567 361.56697

Zinc Chloride 2.00000 63.8433 638.43303

CRYSTALLIZATION CONDITIONS Crystallization of insulin achieved by precipitation of

insoluble zinc-hexamer in aqueous solution, complemented by temperature decrease

Inlet Protein Concentration Amorphous precipitate forms if concentration is too high

Temperature Crystallization begins at ambient temperature (25oC) Cooling to 5oC further decreases solubility

pH Use ammonium acetate buffer to fix pH at approximately

6.1 Acetate anions also improve crystallization

Metal Ion ConcentrationDivalent metal ions (group 15 elements)

mediate intermolecular contactsZinc chloride- soluble in acetic acid, allows

formation of zinc insulin hexamer (pictured)0.08g zinc/g insulin

Other Potential SolventsAddition of 10% (v/v) acetoneIncrease step growth kinetics of insulin

crystals

CRYSTALLIZATION CONDITIONS

PRODUCTION CAPACITY (SUPERPRO)

Insulin (12.78 kg/batch)

WFI (161.09 kg/batch)

Zinc Chloride (2.00 kg/batch)

INPUTS

Acetic Acid (2.22 kg/batch)

Ammonium Acetate (1.13 kg/batch)

OUTPUTS

Acetic Acid (2.22 kg/batch)

Zinc Chloride (2.00 kg/batch)

Insulin (1.28 kg/batch)

Ammonium Acetate (1.13 kg/batch)

WFI (161.09 kg/batch)

Insulin Crystal (11.50 kg/batch)

Total Mass Flow Rate = 176.09 kg/batchTotal Volumetric Flow Rate = 175.37 L/batch

Assumptions:• Working volume is 75% of total tank volume• Safety factor of 15%• REQUIRED TANK SIZE = 270 L = 71 US gal

BATCH-TIME OPTIMIZATION

Complete economic analysis is necessary to find optimal operating throughput (number of batches)

Capital and operating cost optimization for entire process

Equipment considerations to prevent bottlenecks and downtime (batch and continuous units)

0 200 400 600 800 1000 1200 14000

10

20

30

40

50

60

70

80

90

100

Time (minutes)

Cry

stal

liza

tio

n (

%)

Crystallization extent as a function of time for porcine insulin (adapted from Schlichtkrull, 1958)

MATERIALS OF CONSTRUCTION

304 SS 316 SS Carbon Steel

Acetic Acid D-Severe Effect

B-Good C-Fair

Ammonium Acetate

B-Good A-Excellent Unknown

Zinc Chloride B-Good B-Good D-Severe Effect

Steel provides excellent strength, durability, and ease of cleaning/sterilization

Three types of steel investigated for chemical compatibility with chemicals present in the inlet/outlet streams Carbon Steel

Stainless Steel 304

Stainless Steel 316

Type 316 SS chosen as material of construction for crystallization tank

Chemical Compatibility of Various Materials with Inlet Chemicals

COSTING AND SUPPLIERS Unsuccessful attempts to contact vendors

Capital cost estimate using Matches online utility

270 L stainless steel crystallizer = $ 65,378.76 (2009 CAD)

Approximate dimensions assuming H:D ratio of 3 0.5 m diameter x 1.5 m height

Adjusted 2007 USD cost using CEPCI and exchange rate of 0.80 USD/CAD

Total cost of chemicals per batch: approximately $390 CAD/batch ($576.00 with 10% v/v acetone)

Potential suppliers: Solvias Inc. (Fort Lee, NJ), Paul Mueller Co. (Springfield, MI)

SEPARATION AND STERILIZATION Crystal separation is achieved by basket

centrifugation following V-106

Steam-in-place (SIP) sterilization between batches using saturated steam (15 psi, 121°C)

ALTERNATIVE UNIT OPERATIONS Several options available to obtain unique drug types or zinc free insulin:

Gel filtration chromatography Ultrafiltration Diafiltration

These units can successfully purify insulin, though a crystalline product would not be obtained

Other common crystallization procedures include: Evaporative batch crystallization Adiabatic crystallization Continuously seeded crystallization

Wide variety of reagents available and acceptable for insulin crystallization Use of phosphate & citrate buffers Crystallization of monomeric insulin analogs