Experiment #5: Protein Separation Project

Michael EatmonSarah Katen

Nell KeithMonica Sanders

Introduction

Project Description and Goals (1)• Scale-up of chromatography column

used to separate a mixture of bovine hemoglobin and bovine albumin

• Batch process – specified amount of protein loaded on to column for each run

• Required to process 50,000 liters/year of a mixture of 6.0 g/liter albumin and 4.0 g/liter hemoglobin

Project Description and Goals (2)

• Total Capital Investment (TCI), cash flow, and profit of project will be evaluated

• Selling price of hemoglobin is $700 per kg

• Selling price of albumin is $500 per kg

Methods Considered• Method #1: Purchase a single large

column and run batches only on this column – lowest initial investment, but greater up-time and operating costs.

• Method #2: Purchase multiple columns and only run one batch per day per column – highest initial investment, but lowest operating costs.

• Optimum method between these extremes

Theory

Theory (1)• Anion exchange chromatography –

resin is a weak base and attracts negative ions when it is mixed when mixed with a solution at a pH below its pK (~10)

• TRIS-chloride buffer used b/c TRIS is neutral and will not interact with the resin

• Salt dissolved in TRIS buffer to cause proteins to desorb

• Components in stationary phase move down the column more slowly than those that dissolve in the mobile phase

Theory (2)

Image courtesy of http://www.cartage.org.

Theory (3)

•Isoelectric point = pH at which net charge per amino acid is zero•Isoelectric point bovine hemoglobin = 6.8•Isoelectric point bovine albumin = 4.7•Bovine albumin will come out of the column first because it is less charged in the buffer solution and has a lower affinity for the resin.

Theory (4)• Column regenerated between runs by

washing it with NaOH at pH of 14 to remove biological compounds adsorbed to resin.

• Resin stored in 20% ethanol to prevent drying

Theory (5)• Pressure drop monitored to

determine if column is appropriate for scale up

• Column length limited by pressure drop

• Calculated with the Darcy Equation

k

vLp

Theory (6)• Need superficial velocity and Blake-

Kozeny constant to get the pressure drop.

2r

Qv

1150

22pdk

Design Plan

Design Plan (1)• For scale-up, resolution is held

constant

• Resolution of proteins in linear gradient elution ion exchange chromatography:

21

2)(

po

ms duVVg

LDR

Design Plan (2)• To remove the volume terms in this

expression, we define

V

Au

V

QQ

gVG

Design Plan (3)• Substituting into previous equation

gives

21

2)1(

p

ms DQG

DR

Design Plan (4)• For scale-up purposes, the diffusion

coefficient and column void fraction will be the same for the pilot and scaled-up design

2222

2111 pp dQGdQG

Design Plan (5)• As particle size increases, either Q or

dp must decrease while the other is held constant

• The same resin and particle size will be used for the scaled-up design

• This gives:

2

2

1

1

V

Q

V

Q

Pressure Drop Restrictions• The column height can be changed

along with the linear velocity using the normalization between the two in order to meet pressure drop restrictions.

• When the flow rate is kept constant, the pressure drop decreases as column diameter increases

Scale-up Plan (1)• In this experiment, desired elution

flow rate is not given; the desired production is given in throughput

• The scale-up relationship

can still be used as we know that throughput is proportional to elution flow rate

2

2

1

1

V

Q

V

Q

Scale-up Plan (2)• The desired throughput is divided by

the pilot throughput to get a scale-up factor (SUF) that will be used to determine the scaled-up column volume

• With the new volume, a scaled-up column height (L) may be determined using prefabricated column diameters as specified by manufacturers

Optimization• In order to optimize the system,

several diameters may be considered to find the most favorable system that will meet pressure drop restrictions.

• Also, the number of columns and batches per day will be variables in scale-up optimization.

Experimental Plan

Experimental Plan (1)• The major variables in the system

are: – the rate of change of the salt gradient– the final salt concentration within the

column– the gradient elution velocity– the amount of protein loaded into the

column over a given amount of time.

Experimental Variables (1)• First variable - rate of change of the salt

gradient• Depends on elution volumes used• Lower volume gives more rapid rate of

change• Different proteins elute at different salt

concentrations• Too rapid (low volume) – poor or no

separation• Too slow (high volume) – high separation

time

Experimental Variables (2)• Second variable – final salt concentration• Changing the concentration of NaCl

introduced into the system; a higher concentration gives a more rapid rate of change.

• Different proteins elute at different salt concentrations

• Too high – poor or no separation• Too low – high separation time

Experimental Variables (3)• Third variable – gradient elution

velocity

• Too fast – proteins wash out in bulk; poor separation

• Too slow –separation time too slow for industrial processes

Experimental Variables (4)• Fourth variable – amount of protein

loaded into the column over a period of time

• Too much – not enough absorbent surfaces to hold protein – poor separation

• Too little – requires more time to separate desired throughput – inefficient

Apparatus

Operating Equipment• Bovine albumin and bovine

hemoglobin

• Bio-Rad econo column– DEAE Sepharose Fast Flow

• Bio-Rad econo UV monitor

• Bio-Rad gradient former

Operating Procedures• Protein separation column created

• Protein loaded onto column

• Salt gradient added at feed rate

• Salt gradient added at elution rate

• Protein separation measured on chart recorder

• Regenerate column for next run

Operating Variables• Elution rate and salt gradient varied

• Salt gradient volume: 75-100 mL

• Elution rate: 3.0-4.0 mL/min

• pH ≈ 8.2

• Total cycle time recorded

Safety Hazards

• Should be handled in a well ventilated area

• The skin should be rinsed 15 minutes upon exposure to the skin

• Bovine hemoglobin can irritate the upper respiratory tract causing allergic reactions and asthma

Experimental Results

Protein Separation Trial Runs• 9 different trials

• Maximize protein separation as measured by protein peak area ratios

• Minimize time between protein peaks

• Consistency

Successful Trial Run Conditions

Bed Height 14.25 cm

Gradient Mixer Volume

75 mL in each side

Gradient 0 to 0.5 M NaCl in buffer

Feed 2.5 mL loaded at 0.5 mL/min (5min)

Elution 3 mL/min

Detector 2.0 AUFS

Chart Speed 30cm/hr

Elution Time 50 min

Trial Results

• Trial 1: Some separation, protein peaks spread very far apart

• Trial 2: Better separation, but peaks too close together

• Trial 3: Best separation, very small time between elapsed between peaks

• Trial 4-6: Little or no separation, indistinct peaks

• Trial 7-9: Incorrect peak formation, no peaks

Trial #1

Trial #2

Trial #3 (run to be scaled-up)

Hemoglobin/Albumin Peak Ratio

 

peak area of bovine albumin,

cm2

peak area of bovine

hemoglobin, cm2

hemoglobin/albumin ratio

Trial 1 255 119 0.467

Trial 2 198 205 1.035

Trial 3 88 326.5 3.710

Trial 4 18 296 16.444

Trial 5 4 238 59.5

Trial 7 16 172 10.75

Scaled-Up Design

Scale-up Procedure• Scale-up factor (SUF) determined by

dividing desired protein throughput per column by pilot throughput

• Trial #3 (10/5/04) scaled-up

• Assumption of 8-hour workday, 5-day workweek

• Assume 20-year project life; construction in 2004, production begins in 2005

Parameters to be Scaled-Up

Experimental Variable Pilot Value

Bed height (cm) 14.75

Column inner diameter (cm) 0.8

Column volume (cm3) 7.41

Protein throughput (g) 0.025

Final salt concentration (M) 0.5

Gradient volumes (mL) 75

Elution rate (mL/min) 3.0

Process to be Scaled-up• Equilibration with 20 mM TRIS buffer (pH 8.2), 5 bed

volumes in 20 minutes.

• Load protein

• Elute protein to final column concentration of 0.5 M NaCl in 20 mM TRIS, assuming same residence time as pilot scale

• Regenerate bed by flooding with:

a)  3 bed volumes 1.0 M NaCl in 15 minutes

b)  5 bed volumes 1.0 NaOH in 20 minutes c)  3 bed volumes deionized water in 15

minutes d)  3 bed volumes 20 mM TRIS in 15 minutes

• Repeat steps 3 and 4 for each run.

• Flood column with 20 % ethanol (1 bed volume) in 20 minutes for storage.

Scale-up Procedure (1)• Desired daily throughput divided by

arbitrary number of runs per day

• Column SUF determined from throughput and applied to pilot column volume to determine scaled-up volume

• Prefabricated column specifications used to determine bed height for given diameters

Scale-up Procedure (2)• Scaled-up specs. used to determine

superficial velocity and then pressure drop across column, to ensure compliance with column pressure rating

• Total cycle time used to determine the number of possible cycles per day

• Cycles per day divided by cycles per column per day to determine number of columns for scaled-up design

Scale-up Procedure (3)• Arbitrary number of runs from initial

step varied iteratively in order to minimize number of columns for each set of specs.

• Economic optimum at point of lowest equipment cost, as further calculations are based on total delivered equipment costs

Scaled-up Process• 4 columns

- 35 cm x 50 cm

- P<2 bar

• Bed volume of 48 L

• Three runs per column per day

• Daily throughput of 2 kg protein

• Gradient volumes per run of 490 L

• Elution rate of 20 L/min

Process Equipment• Equipment sized based on scaled-up

specifications

• Components include chromatography columns, gradient and buffer tanks, fluid pumps, mixers, and tubing

• Equipment priced at lowest possible cost while meeting design requirements

Purchased Equipment Cost and Total Capital Investment

• Equipment prices determined from up-to-date online catalogues

• Total purchased equipment cost: $65,000

• Total Capital Investment (TCI) determined by method of percentage of delivered equipment cost

• TCI: $380,000

Product Costs and Profitability• Annual Product Cost (APC) is

summation of all yearly raw materials, utilities, labor, and fixed production charges

• APC (2005): $480,000

• APC per kg/feed: $960

• Revenue from sales less APC is annual cash flow

• Return on Investment (ROI): 390%; above standard minimum of 10% (depreciation neglected)

Sources of Error• Approximately 5% error inherent in

calculation of SUF

• Scale-up parameters determined by arbitrary assumptions—assigned an approximate 10% error

• Accuracy in equipment pricing lends itself to TCI accuracy within 10%

• Error in APC neglected

Comparison to Literature Values

Comparison to Literature Values (1)

• Ratio of peak areas of experimental data

71.388

5.3262

2

minexp

cm

cm

A

Ar

albu

hemoglobin

Comparison to Literature Values (2)

• Ratio of absorbance of hemoglobin to that of albumin times the concentration ratio

91.4594.0

491.2

/6

/4

minminexp

Lg

Lg

Abs

Abs

C

Cr

albu

hemoglobin

albu

hemoglobin

Comparison to Literature Values (3)

• Experimental Error Calculation

%7.32%10080.2

80.271.3

References• Laboratory Operating Manual. Design Lab, CH E

4262, 2004.

• Harrison, Roger G., Paul Todd, Scott R. Rudge, Demetri Petrides. Bioseparations Science and Engineering. New York: Oxford University Press, 2003.

• Peters, Max S., Klaus D Timmerhaus, and Ronald E. West. Plant Design and Economics for Chemical Engineers, 5th ed. New York: McGraw-Hill Companies, Inc., 2003

• Welty, James R., Charles E. Wicks, Robert E. Wilson, and Gregory Rorrer. Fundamentals of Momentum, Mass, and Heat Transfer, 4th ed. New York: John Wiley & Sons, Inc., 2001.