Economic Analysis of Recombinant Human Lysozyme Economic Analysis of Recombinant Human Lysozyme and Hen Eggand Hen Egg--white Lysozyme Purificationwhite Lysozyme Purification

March 8, 2008

Lisa Wilken and Zivko NikolovBiological & Agricultural EngineeringTexas A&M University

Applications Applications

Recombinant Human Lysozyme from Rice◦ Cell lysis: in culture protein extraction◦ Baby formula additive, oral rehydration solution

Hen Egg-white Lysozyme◦ Food Processing

Lysozyme accepted by FAO/WHO in 1993 under GMP

◦ Have you consumed lysozyme lately?Common preservative for wine and cheese industry

1.5 mg per

8 oz. 7 mg

in 20 g slice

www.invitria.com

PurposePurpose

Determine whether human lysozyme could be economically competitive to hen egg-white lysozyme◦ 1 ton of purified lysozyme/yr◦ >90% purity

SuperPro Designer ® (Intelligen Inc.)◦ Tool for process development and to guide research◦ Incorporate lab data and processing knowledge into simulation◦ Simulate effect of variations in material costs and process

parameters

Process DiagramsProcess Diagrams

Extraction

IEX

Transgenic Rice FlourBuffer

Clarify Residuals

Rice Impurities

Hen Egg-white Lysozyme

Precipitation

Liquid Egg-whiteWater

IEX

Clarify Ovomucin Precipitate

Egg Impurities

Salts

SprayDry

SprayDry

Salts

Human Lysozyme

Diafiltration Diafiltration

Processing ParametersProcessing Parameters

Human Lysozyme Extraction1

◦ Maximize lysozyme recovery◦ Maximize lysozyme to native protein ratio

Cation Exchange Chromatography◦ Binding capacity

Minimize column size and cost

◦ PurityMinimize # of purification steps

◦ YieldReduce raw material requirements Reduce number of batches and processing time

1Wilken and Nikolov. 2006, 22(3) 745-752.

P-2 / V-101

Ovomucin Precipitation

Vent

pH=6

P-4 / DS-101

Centrifugation

EW/PPT

Ovomucin Ppt.

P-5 / V-103

Blending / StorageNaPhosphate

NaCl

Sodium Phosphate/Sodium Cloride Added

P-6 / DE-101

Dead-End Filtration

S-105

Clarified EW

Waste

Ovomucin Precipitation & Clarification

P-7 / C-101

IEX Chromatography

Load

WashEluant

NaOH (0.5M)

Liquid Waste-1

Hen Egg-White Lysozyme Purification

P-8 / DF-101

Diafiltration

HewLZ Pool

Water for DF

Liquid Waste-2

Egg-white

Water

P-1 / SDR-101

Spray Drying

Conc. HewLZ

AirProduct

Air Out

Phosphoric Acid

DiafiltrationDrying

IEX Capture3.2 kg/batch

998 kg/batch

2978 L/batch

Precipitation Solid-liquid Separation

Cost Estimation: Raw MaterialsCost Estimation: Raw Materials

$0.33/kg liquid egg white $1/kg brown rice◦ Calculated from production cost

$750/acre transgenic rice (assumed 2x cost of nt rice)$375/acre non-transgenic rice (USDA)

Brown rice 80% of rough riceCost calculated with range of yields (± 20%)

Why cost difference?Identity preservationCrop isolation

Grow rice in Kansas?

$/kg brown ricelow yield average high yield

0.43 0.34 0.29

Raw MaterialsRaw Materials

How many acres of rice are needed for 1 ton human lysozyme/year?◦ 8-12 kg lysozyme/acre if 2200-3300 kg rice/acre yield1

◦ 85-130 acres

How many eggs does it take to produce 1 ton of hen egg-white lysozyme/yr? How many chickens?◦ 10 million eggs or 38,400 chickens◦ 0.5 acre required2

1 USDA Rice Yearbook 89001, ERS2 United Egg Producers Certified Welfare Guidelines

1x107

eggs!

Cost Estimation Cost Estimation

Equipment◦ User-defined model based on quotes◦ Built-in model

Capital investment◦ Based on equipment cost calculated using SuperPro◦ Lang factor of 3

Reagents◦ ICIS chemical cost

Human LZ HewLZEquipment (FOB) Cost 1435 1233Direct Fixed Capital (DFC) 4305 3699Working capital ( 3% DFC) 129 111Start-up cost (5% DFC) 215 185Total Capital Investment (DFC+WC+Startup) 4649 3995

$1000/yr

Cost ComparisonCost Comparison

Direct vs. Indirect Manufacturing Costs

Indirect Manufacturing Cost Human LZ HewLZMaintenance (3% DFC for this project) 0.13 0.11Insurance (1%) & property (2%) taxes (3% DFC) 0.13 0.11Administration (20% of operating labor) 0.14 0.14Depreciation (10yrs) 0.42 0.36Total Indirect Manufacturing Cost ($/g) 0.8 0.7

$/g

Direct Manufacturing Cost Human LZ HewLZRice flour or Egg-white 0.27 0.11Reagents (buffers, salts, all water) 0.16 0.11Consumables (membranes, filters, resins) 0.12 0.17Operating labor with benefits (12 @ $30/h) 0.72 0.72Supervision (20% of operating labor) 0.14 0.14Lab, QC/QA (15% of operating labor) 0.11 0.11Operating supplies (10% of labor + lab personnel) 0.10 0.10Waste disposal 0.07 0.03Total Direct Manufacturing Cost ($/g) 1.7 1.5

$/g

Section Cost: % of Total Production CostSection Cost: % of Total Production Cost

IEX consistently most expensive!Evaluate major cost contributors to this section

18%

5%

13%

37%

22%

5% Grinding

Extraction

Solid‐Liquid Separation

IEX 

Diafiltration

Drying

20%

16%

35%

19%

10% Precipitation

Solid‐Liquid Separation

IEX

Diafiltration

Drying

Human Lysozyme

Hen Egg-white Lysozyme

Cost ContributorsCost Contributors

IEX Cost Distribution

Major Cost Contributors◦ Facility-related◦ Labor

Human Lysozyme Hen Egg-white Lysozyme

Considerations & Process ImprovementsConsiderations & Process Improvements

Economy of scale◦ Most important in preliminary sensitivity analysis

Higher purity lysozyme for other applications◦ Simulated an alternative human lysozyme process to give higher

final product purity 8% increase in annual operating cost but potentially higher value product

ConsiderationsConsiderations

Value of byproducts◦ Rice flour residuals for biofuels◦ Egg white protein

Cost per unit similar for both lysozymes ◦ Human lysozyme activity 3x activity of hen egg-white lysozyme◦ Application will dictate product specs, throughput, and cost

AcknowledgementsAcknowledgements

Ventria Biosciences, Dr. Ning HuangDr. Zivko Nikolov