Chapter 11: Product Recovery and Purification

David Shonnard Department of Chemical Engineering Michigan Technological University

David R. Shonnard

Michigan Technological University

1

Presentation Outline:
Overview of Bioseparations Separation of Insoluble Products Primary Isolation / Concentration of Product Purification / Removal of Contaminant Materials Product Preparation

l l l l l

David R. Shonnard

Michigan Technological University

2

Introduction to Bioseparations
Characteristics of Bioseparations vs Chemical Separations
Characteristics
Environment Concentration Range Temperature Sensitivity

Biochemical
Aqueous Media v. Dilute Product Product Vulnerable

Chemical
Organic Media Concentrated Product Product Not Vulnerable

Traditional chemical separations are unsuitable or must be augmented

David R. Shonnard

Michigan Technological University

3

Biochemical Separations Technologies
Technologies

“Bioprocess Engineering: Basic Concepts” Shuler and Kargi, Prentice Hall, 2002

David R. Shonnard

Michigan Technological University

4

Figure 11.2 Major Steps in Separating a Protein Product

Fermentation Filtration Centrifugation Coagulation / Flocculation Ultrasonic Vibrators French Press Bead or Ball Mills Ultracentrifugation Ultrafiltration

1. Separation of Insoluble Products, Product Concentration (0.2 - 2.0%)

Cell Removal and Concentration

Cell Disruption

Removal of Cell Debris

David R. Shonnard

Michigan Technological University

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Figure 11.2 Major Steps in Separating a Protein Product

2. Product Isolation, (removal of water) (1 - 10%)

Protein Precipitation or Aqueous TwoPhase Extraction

Other methods for product isolation: Liquid-liquid extraction Adsorption

Ultrafiltration

David R. Shonnard

Michigan Technological University

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Figure 11.2 Major Steps in Separating a Protein Product

Chromatographic Purification 3. Purification / Removal of Contaminant Chemicals, (50 - 80%)

Solvent Precipitation

Other methods for product purification: Reverse Osmosis Cross-flow Ultrafiltration Electrophoresis Electrodialysis

Dialysis

David R. Shonnard

Michigan Technological University

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Figure 11.2 Major Steps in Separating a Protein Product

4. Product Preparation, (90 - 100%)

Lyophilization

Other methods: Crystallization

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Michigan Technological University

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1. Removal of Insoluble Products Rotary Vacuum Filtration

• coagulation agents/ (filter aids) added • vacuum applied to rotating drum (∆P)

Cell Solution

“Bioprocess Engineering: Basic Concepts” Shuler and Kargi, Prentice Hall, 2002

David R. Shonnard

Michigan Technological University

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1. Removal of Insoluble Products Rotary Vacuum Filtration

Filter area

Volume filtered

g c ∆P A dV = dt (rm + rc ) µ

Viscosity of filtrate (water) Filter cake resistance

Filter medium resistance (a constant)

CV rc = α A

Note: rc increases with the volume filtered, V

C = wt. of cells per volume filtrate (g cells/L) α = average specific resistance of filter cake
David R. Shonnard Michigan Technological University 10

1. Removal of Insoluble Products Rotary Vacuum Filtration
Integrate Filter Equation: V=0 at t=0.
V2 + where Vo = rm 2VV o = Kt
Ruth Equation

α C

A
2

! 2 A $ K = # & ∆ P gc "α Cµ%

! kg •m 1 2 # s gc = # N # "
N = Newton

$ & & & %

David R. Shonnard

Michigan Technological University

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1. Removal of Insoluble Products Rotary Vacuum Filtration

Rearrange Ruth Equation

1 t = (V + 2Vo ) V K ! 2 A2 $ α = slope • # &∆P g c #C µ & % " 1 = K

! 2 A2 $ •# #C µ & & ∆P g c " % K K αC Vo = y - intercept • ' rm = y - intercept • 2 2 A
David R. Shonnard Michigan Technological University 12

“Bioprocess Engineering: Basic Concepts” Shuler and Kargi, Prentice Hall, 2002

Rotary Vacuum Filtration

Effect of pH and time on volume filtered

“Bioprocess Engineering: Basic Concepts” Shuler and Kargi, Prentice Hall, 2002

David R. Shonnard

Michigan Technological University

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Rotary Vacuum Filtration

Effect of filter aid and time on volume filtered
Typical Filter Conditions • pH = 3.6 • 2% - 3% filter aid • heat treatment, T=80˚C
“Bioprocess Engineering: Basic Concepts” Shuler and Kargi, Prentice Hall, 2002

David R. Shonnard

Michigan Technological University

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1. Removal of Insoluble Products Rotary Vacuum Filtration; Example 11.1
Yeast Cell suspension Filtration
Rotary Vacuum Filtration….
0.12 0.10 0.08 0.06 0.04 0.02 0.00 0 200 400 600 800 1000 1200 y = 6.7E-05x + 0.0272 R2 = 0.9906

V (Liters of Filtrate)

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Michigan Technological University

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1. Removal of Insoluble Products Rotary Vacuum Filtration; Example 11.1
slope = 1 = 6.7 x10 −5 (min/L2 ) K ! 2 A2 $ 1 4 2 & K= = 1.5 x10 (L /min) = # ∆P g c −5 # & 6.7 x10 "α C µ %
* not 9.8 kgm/(kgf s2) as in the book

! 2 A2 $ α= # #KCµ& & ∆P g c " %

N $! kg m $ ! 2(.28 m 2 ) 2 # 2.3x10 − 4 2 &#1 2 & m %" s • N % " = 2 3 2 −3 ! $ ! $ ! L 10 m kg $! 4 3 kg $! 1 min $ # & # & x x 1 . 5 10 19 . 2 2 . 9 10 &# & 3 &# # &# L & # min m m s 60 s • % " % " % " " %" % = 2.59 m kg
* 1,920 as in the book
16

David R. Shonnard

Michigan Technological University

1. Removal of Insoluble Products Microfiltration/Ultrafiltration
• for particle size range ~ 10-9 to 10-5 m = dp • purpose → to concentrate a cell suspension → to recover dissolved solutes / proteins
Cross-Flow Filtration “Retentate” (concentrated suspension) Filtrate or “Permeate” (dissolved solutes / proteins)

Feed Tank

Membrane module

pump
David R. Shonnard

Microfiltration membrane
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1. Removal of Insoluble Products Microfiltration for Removal of Cells
• Mass balance model for separation of cells
(cells are retained in the feed tank)
qR,CR Retentate
Chandrasekaran, R., MS Thesis, Dept. of Chemical Engineering MTU

MF Membrane Permeate
Mass Balance Assumptions 1. Feed tank is well mixed. 2. Permeate tank is well mixed. 3. Volume of fluid in MF cassette is negligible. 4. Densities of each stream are equal.

qP, CP

Feed Tank

VF(t) MF Cassette Feed qF, CF VP(t)

Permeate Tank

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Michigan Technological University

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1. Removal of Insoluble Products Microfiltration for Removal of Cells
Chandrasekaran, R., MS Thesis, Dept. of Chemical Engineering MTU

Feed Tank

A total mass balance assuming constant stream densities leads to equation [1] for the change in feed tank volume,
V F (t ) .
dV F (t ) = q R − q F = − q P …………………………………………[1] dt

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Michigan Technological University

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1. Removal of Insoluble Products Microfiltration for Removal of Cells
And similarly for entering and exit streams for the membrane cassette, where
qF , qR ,

and

qP

are the volumetric flow rates of

the feed, retentate, and permeate streams.

q F = q R + q P ……………………………………………………..[2]
Chandrasekaran, R., MS Thesis, Dept. of Chemical Engineering MTU

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Michigan Technological University

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1. Removal of Insoluble Products Microfiltration for Removal of Cells
A cell mass balance on the feed tank results in equation [3], where
CF , CR ,

and

CP

are the concentrations of the cells in the

feed, retentate, and permeate streams.
Chandrasekaran, R., MS Thesis, Dept. of Chemical Engineering MTU

d (C F VF (t )) = q R C R − q F C F ……………………………………[3] dt

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Michigan Technological University

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1. Removal of Insoluble Products Microfiltration for Removal of Cells

A cell mass balance on the cassette results in equation [4],
q F C F = q R C R + q P C P ……………………………………….….[4]

For a perfectly retained cell: C P = 0 , and equation [4] becomes [5]
q R C R = q F C F ………………………………………….…..….[5]

Chandrasekaran, R., MS Thesis, Dept. of Chemical Engineering MTU

David R. Shonnard

Michigan Technological University

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1. Removal of Insoluble Products Microfiltration for Removal of Cells

Substituting [5] into [3] (for a perfectly retained cell)

Chandrasekaran, R., MS Thesis, Dept. of Chemical Engineering MTU

d d (C F V F (t )) = m F = q R C R − q F C F dt dt

= qF CF − qF CF = 0
d m F = 0 where m F is mass of cells in feed tank ( m F = C F VF (t ) )….[7] dt

David R. Shonnard

Michigan Technological University

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1. Removal of Insoluble Products Microfiltration for Removal of Cells
Chandrasekaran, R., MS Thesis, Dept. of Chemical Engineering MTU

mFo mF

Integrating; ( dm F = ( 0dt ' m F = Constant

t

At t = 0, m F = m F ( m F is the initial mass of cells in the feed tank)
0 0

m F = m F0 for all time t “perfectly retained cell”…………[8]

David R. Shonnard

Michigan Technological University

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1. Removal of Insoluble Products Microfiltration for Removal of Cells

Cell Concentration, CF (t )

Chandrasekaran, R., MS Thesis, Dept. of Chemical Engineering MTU

(

d (CFVF ( t )) = mFo VF (t )

( 0 dt
= mFo VFo − qP t
CF

CF VF (t ) = constant = mFo CF =

t
David R. Shonnard Michigan Technological University 25

1. Removal of Insoluble Products Microfiltration for Removal of Cells
Perfectly Permeating Cell (or Protein)
m F = − q P C F0 t + Constant……………………….……[11]
At

t = 0, m F = m F0 '

Constant

= m F0

Chandrasekaran, R., MS Thesis, Dept. of Chemical Engineering MTU

m (t ) m − qP C Fot C F (t ) = F = Fo V (t ) Vo − q P t note that C Fo = mFo Vo

CF

m q mFo − q P Fo t mFo (1 − P t ) Vo Vo m C F (t ) = = = Fo = C Fo q Vo − q P t Vo Vo (1 − P t ) Vo
David R. Shonnard Michigan Technological University

mF t
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1. Removal of Insoluble Products Microfiltration for Removal of Cells
Partially Retained Cell (or Protein)
Some fraction (θ) of the cells (or protein) is of a size that is retained and (1-θ) permeates.
Chandrasekaran, R., MS Thesis, Dept. of Chemical Engineering MTU

(1 − θ ) ) CF where CF is Concentration Factor V VFo CF = Fo = VF (t ) VFo − qP t mF = mFo (θ + C F = C Fo (CF θ + (1 − θ ))
CF
David R. Shonnard Michigan Technological University

CF

mF

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Microfiltration of Skim Milk to Separate Casein Protein (CP) from Whey Protein (WP)
Protein mass versus concentration factor, CF = VFo/VF
Chandrasekaran, R., MS Thesis, Dept. of Chemical Engineering MTU

David R. Shonnard

Michigan Technological University

28

Microfiltration of Skim Milk to Separate Casein Protein (CP) from Whey Protein (WP)
Protein concentration versus concentration factor, CF = VFo/VF
Chandrasekaran, R., MS Thesis, Dept. of Chemical Engineering MTU

David R. Shonnard

Michigan Technological University

29

Microfiltration of Skim Milk to Separate Casein Protein (CP) from Whey Protein (WP)
Comparison of model with experimental data
Chandrasekaran, R., MS Thesis, Dept. of Chemical Engineering MTU

David R. Shonnard

Michigan Technological University

30

1. Removal of Insoluble Products Microfiltration/Ultrafiltration
Water (Permeate) Velocity Equation
CW

J = KP ( ∆PM - σ∆ π ) where J = water (permeate) velocity K P = membrane permeability ∆ PM = pressure drop across membrane
∆PM
Retentate Flow

σ = "reflection coefficient" ∆ π = osmotic pressure (RTC W )
“Bioprocess Engineering: Basic Concepts” Shuler and Kargi, Prentice Hall, 2002

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Michigan Technological University

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1. Removal of Insoluble Products Microfiltration/Ultrafiltration
Concentration Polarization - relating CW to CB In the liquid film;

dC J =D dx x =δ x=0 integrating J= D CW CB

C = CW C = CB

Gel Formation When J and/or CB are high enough, a gel layer will form at the membrane surface, causing an additional resistance (RG) to solute flux, J.

δ

ln

where D is the diffusivity of solute in the film
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1. Removal of Insoluble Products Microfiltration/Ultrafiltration
Permeate Flow Rate
“Bioprocess Engineering: Basic Concepts” Shuler and Kargi, Prentice Hall, 2002

∆ PM = Pi J =

1 (Pi - Po ) 2

∆ PM RG + RM

Gel resistance Membrane resistance

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1. Removal of Insoluble Products Microfiltration/Ultrafiltration
Permeate Flow Rate

@ constant ∆PM
RM dominant

@ constant TFR

Flux, J
RG dominant but decreasing

Flux, J

Osmotic Pressure Effect Increasing Increasing protein concentration

Tangential Flow Rate (TFR)

∆P Across Membrane (∆PM)

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Michigan Technological University

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1. Removal of Insoluble Products Microfiltration/Ultrafiltration
Concentration Polarization - relating CW to CB Example of Protein Ultrafiltration;

J = 1.3x10-3 cm / sec D = 9.5x10-7 cm2 / sec (protein diffusivity)

δ = 180x10-4 cm
CW J = ln δ CB CW = 1.3 CB
David R. Shonnard

D

9.5x10 cm / sec C W ' 1.3x10 cm / sec = ln 180x10-4 cm CB
-3

-7

2

or CW is 30% > than C B

Michigan Technological University

35

1. Removal of Insoluble Products Microfiltration/Ultrafiltration
Microfiltration Design - time for filtration

dV = -A J dt V = volume of solution remaining to be filtered A = membrane filter area if we assume no concentration polarization, CW ≈ CB dV = - A K P ( ∆P - σ RTCB ) dt for total reflection of solute, σ = 1 and n = C BV and is constant, where n is total solute mass (cells) [RTn / ∆ P $ dV = - A K P ∆P ! 1" % V dt
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1. Removal of Insoluble Products Microfiltration/Ultrafiltration
Microfiltration Design - time for filtration (cont.)

dV ! [RTn / ∆ P $ = - A K P ∆P 1" % V dt at t = 0 V = Vo (initial volume of solution) integrating ) ,/ 1 ! R T n $ ln! Vo - RTn / ∆P $ 2 t = + V) + (V 3 .0 o " % " % ∆P V - RTn / ∆ P 4 * A K P ∆ P -1 RTn << (Vo - V) often ∆P ) , 1 Time to filter from Vo to V. t ≈ + . (Vo - V) * A K P ∆P David R. Shonnard Michigan Technological University 37

1. Removal of Insoluble Products Microfiltration/Ultrafiltration
Microfiltration Design - Example, Cell Microfiltration

Vo = 1000 liters, Xo = 1 g dcw / L

A = 10 m2 concentration to X = 10 g dcw / L

K P ∆P = initial water flux = 5.7x10-4 cm / sec V = Vo Xo ! 1 g dcw / L $ = 1000 L # & = 100 L " 10 g dcw / L % X

) , ! 103 cm3 $ 1 t = + & . (1000 -100)L# 2 2 2 -4 )(100 cm / m ) (5.7x10 cm / sec) L % " (10 m * = 1.58x10 sec = 4.4 hours
38
4

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Michigan Technological University

“Bioprocess Engineering: Basic Concepts” Shuler and Kargi, Prentice Hall, 2002

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Michigan Technological University

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1. Removal of Insoluble Products Microfiltration/Ultrafiltration
Modes of Operation 1. Concentration
Retentate Permeate

2. One-Pass

Retentate Permeate
40

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Michigan Technological University

1. Removal of Insoluble Products Microfiltration/Ultrafiltration
Modes of Operation 3. Total Recycle Mode - membrane system characterization

Retentate

Permeate

Post-Processing:
Microfiltration of cells is often followed by conventional filtration of retentate or centrifugation. Then, cell disruption for recovery of intracellular proteins occurs.
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1. Removal of Insoluble Products Centrifugation
Continuous Centrifuges
Cell-free liquid exit
“Biochemical Engineering” Blanch and Clark, Marcel Dekker, 1997

Cell solids

Cell solids exit

Tubular
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Bowl or Disk
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1. Removal of Insoluble Products Tubular Centrifugation
FD = drag force on cell = 3πµDPU OC
FB L FA FD

r

1 gc 1 gc

FA = centrifugal force on cell = FB = bouyancy force on cell = FD = FA - FB

π
6

3 DP ρ P rω 2

π
6

3 DP ρ f rω 2

1 gc

UOC

3πµDPU OC = U OC =

π

6 2 rω 2 DP (ρ P − ρ f ) 18µ

3 DP ( ρ P − ρ f ) rω 2

y

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Michigan Technological University

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1. Removal of Insoluble Products Tubular Centrifugation
Design Equations

Radial Travel Time = Axial Travel Time y Vc = Uoc Fc Vc = centrifuge liquid volume; Solving for Fc ; Fc = 2 Uo Σ settling velocity under gravity gD 2 P ( ρ P − ρf ) where Uo = 18µ Fc = volumetric flow rate

2πL ω 2 ! 3 2 1 2 $ Σ = r2 + r1 % " g 4 4
2 Fc is proportional to L and r2

David R. Shonnard

Michigan Technological University

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1. Removal of Insoluble Products“Biochemical Engineering” Blanch and Clark, Cell Disruption ; 11.3 Marcel Dekker, 1997
If the desired product is intracellular, an effective method to break open the cell wall is needed in order to release the products.

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45

1. Removal of Insoluble Products Cell Disruption Equipment
Exposure of cells to high liquid shear rates by passing cells through a restricted orifice under high pressure
“Biochemical Engineering” Blanch and Clark, Marcel Dekker, 1997

rod for valve adjustment

valve valve seat impact ring handwheel

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1. Removal of Insoluble Products Cell Disruption Equipment
Rapid agitation of a microbial cell suspension with glass beads or similar abrasives
circulating pump temperature jacket shaft

variable v-belt

“Biochemical Engineering” Blanch and Clark, Marcel Dekker, 1997

agitator drive motor disk handwheel
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David R. Shonnard

1. Removal of Insoluble Products “Biochemical Engineering” and Clark, Cell Disruption Equipment Blanch Marcel Dekker, 1997
Problem 6.1 Blanch and Clark textbook Protein release from yeast using disruption by an industrial homogenizer
Protein release depends upon the pressure, P, and number of recycle passes, N

Design Equation ! Rm $ c log# & = KNP "Rm - R % R m = maximum protein conc. (mg / L) R = protein conc. (mg / L) K = constant C = constant
48

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1. Removal of Insoluble Products Cell Disruption Equipment
Problem 6.1 Blanch and Clark textbook (cont.)

Determine K and C slope = KP or ln(slope) = ln(K) + C ln(P)
c

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2. Primary Isolation/Concentration of Product: 11.4
Separation Objectives
• Remove water from fermentation broth • Dilute solute (product) → more concentrated solute • Often these steps concentrate chemically similar byproducts (other proteins / biomolecules) Separation Methods A. Extraction (liquid-liquid) B. Adsorption C. Precipitation

not very selective for desired product

None the less, these methods are often applied prior to purification
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2. Primary Isolation/Concentration of Product: Liquid-Liquid Extraction
Liquid-liquid extraction is commonly used, especially in antibiotic fermentations to recover product from broth. Features of liquid extractant 1. nontoxic 2. inexpensive 3. highly selective toward the product 4. immiscible with the fermentation broth Other Applications 1. removal of inhibitory fermentation products (ethanol and acetone - butanol).
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2. Primary Isolation/Concentration of Product: Liquid-Liquid Extraction
Liquid-liquid extraction is commonly used, especially in antibiotic fermentations to recover products from fermentation broth
Fermentation broth + penicillin low pH Fermentation broth penicillin Buffered phosphate Aqueous solution Butyl acetate + penicillin neutral pH Butyl acetate

Butyl acetate solvent

Purified aqueous Solution with penicillin

drying step penicillin 52

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2. Primary Isolation/Concentration of Product: Liquid-Liquid Extraction - Equilibrium
Liquid-liquid extraction takes advantage of solute equilibrium partitioning between the fermentation broth (heavy, H) phase and a light (L) extractant phase.

Y K D = , where K D is a distribution coefficient X Y is the concentration, mass or mole fraction of solute in the light phase X is the concentration, mass or mole fraction of solute in the heavy phase
53

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KD

Y = X

Partitioning is a function of pH for many solutes Solvent is Amylacetate

“Bioprocess Engineering: Basic Concepts” Shuler and Kargi, Prentice Hall, 2002

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2. Primary Isolation/Concentration of Product: Liquid-Liquid Extraction
Mass balance on a single equilibrium stage Assumptions: dilute solute and immiscible phases (negligible change in H and L) and constant KD.

H ( X o − X 1 ) = LY1 or Y1 Since K D = , X1

X1 = X o −

L Y1 H

LK D X1 X1 = X o − H

1 1 X1 or = = X o 1 + ( LK D / H ) 1 + E
“Bioprocess Engineering: Basic Concepts” Shuler and Kargi, Prentice Hall, 2002

LK D where E = is the extraction factor Ho
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2. Primary Isolation/Concentration of Product: Liquid-Liquid Extraction
Mass balance on a multiple equilibrium stages Assumptions: dilute solute and immiscible phases (negligible change in H and L) and constant KD.

“Bioprocess Engineering: Basic Concepts” Shuler and Kargi, Prentice Hall, 2002

Stage N, H ( X N −1 − X N ) = L(YN − YN +1 ) or X N −1 = X N + Since K D = YN , XN X N −1 = X N +

L YN H

LK D X N , or X N −1 = (1 + E ) X N H
56

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2. Primary Isolation/Concentration of Product: Liquid-Liquid Extraction
Stage N - 1, H ( X N − 2 − X N −1 ) = L(YN −1 − YN ) or X N − 2 = X N −1 + X N −2 X N −2 X N −2 L Y Y (YN −1 − YN ), Since K D = N −1 = N , H X N −1 X N

LK D = X N −1 + ( X N −1 − X N ), with X N −1 = (1 + E ) X N H LK D = X N −1 + ( X N −1 − X N ) = (1 + E ) X N −1 − EX N H = (1 + E )(1 + E ) X N − EX N = (1 + E ) 2 X N − EX N

X N − 2 = (1 + E + E 2 ) X N ! E N +! − 1 $ All Stags, X o = # # E −1 & &X N " %
David R. Shonnard

see Figure 11.9
57

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2. Primary Isolation/Concentration of Product: Liquid-Liquid Extraction - Figure 11.9

Relates XN/Xo to E and Number of Stages , N.

“Bioprocess Engineering: Basic Concepts” Shuler and Kargi, Prentice Hall, 2002

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2. Primary Isolation/Concentration of Product: Liquid-Liquid Extraction - Figure 11.9
Example 11.2 Penicillin Extraction using Isoamylacetate L = isoamylacetate flow rate = 10 L/min H = aqueous broth flow rate = 100 L/min KD = 50, Xo = 20 g/L, XN = .1 g/L How many stages are required to achieve this separation? Solution: XN / Xo = 0.1/20 = .005 E = LKD/H = (10)(50)/100 = 5 From Figure 11.9, we see that the required number is stages is between 3 and 4, call it 4 equilibrium stages.
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2. Primary Isolation/Concentration of Product: Liquid-Liquid Extraction - Figure 11.9

“Bioprocess Engineering: Basic Concepts” Shuler and Kargi, Prentice Hall, 2002

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2. Primary Isolation/Concentration of Product: Liquid-Liquid Extraction - Equipment
Podbielniak centrifugal extractor The separation is very rapid, allowing for short residence times which benefits unstable products (especially pHsensitive antibiotics.
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“Bioprocess Engineering: Basic Concepts” Shuler and Kargi, Prentice Hall, 2002

2. Primary Isolation/Concentration of Product: Precipitation
A very common first step after cell disruption for recovery of intracellular proteins. Water-protein interactions are key to understanding protein precipitation / solubility in water. Salting-Out addition of (NH4)2SO4 or Na2SO4 up to high concentrations → 1 to 3 Molar!
COO- -- NH4+

protein
NH4+ -- SO42David R. Shonnard

salts exclude water from the surface leading to protein-protein interactions and precipitation
62

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2. Primary Isolation/Concentration of Product: Precipitation
Protein solubility is a function of ionic strength (salt concentration).

!S$ ' & = − K I log# S #S & " o% S = protein solubility (g/L) S o = protein solubility at 0 ionic sstrength, (g/L)
' KS = a salting out constant (moles/L)

(a function of pH and temperature) 1 I = ionic stsrength = 5 Ci Z i2 (mole/L) 2 Ci = molar concentration of salt ion (mole/L) Z i = charge on salt ion
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2. Primary Isolation/Concentration of Product: Precipitation
Organic Solvent Addition can also reduce protein-water interactions and promote proteinprotein interactions leading to precipitation. Isoelectric Precipitation at the pH of the isoelectric point, a protein is uncharged, reducing protein-water interactions which leads to precipitation. Warning: extremes in pH may denature the protein product.

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64

2. Primary Isolation/Concentration of Product: Isoelectric Precipitation
Belter, Cussler & Hu, 1988
Effects of pH on the charge of protein functional groups NH2 + H+ → NH3+

COOH → COO- + H+

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3. Product Purification /Contaminant Removal:
Contaminants often remain with product after primary isolation. Chromatography: is the most important separation method for biochemical products. Basic Concepts: 1. Separation is based on differential affinities of solutes toward a solid adsorbent material.

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3. Product Purification /Contaminant Removal: (cont.)
2. Different kinds of affinity

*

→ electric charge … ion exchange chromatography → van der Waals force … adsorption chromatography → solubility in liquid … liquid-liquid partitioning chromatog. → solute size/diffusion … gel filtration chromatography → receptor - ligand … affinity chromatography → hydrophobic interactions … hydrophobic chromatography most common usage

* *

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Michigan Technological University

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3. Product Purification /Contaminant Removal: Adsorption - 11.4.4
Definition: the removal of selected chemicals from a mobile fluid phase into an immobile solid phase. Adsorbents: solid materials to which the chemicals (solutes, adsorbates) adhere. These are the immobile phase. Examples: activated carbon ion exchange resins alumina silica gel other gels: dextran or agarose

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3. Product Purification /Contaminant Removal: Adsorption - 11.4.4 (cont.) “Bioprocess Engineering:
Fixed-Bed Adsorption 3 Zones
Saturated zone, solute is present in both fluid and solid at maximum concentration. Adsorption zone, concentrations of solute in fluid & solid are in transition. Virgin zone, concentrations near zero.
David R. Shonnard Michigan Technological University 69
Basic Concepts” Shuler and Kargi, Prentice Hall, 2002

3. Product Purification /Contaminant Removal: Adsorption Equilibrium
Freundlich Isotherm: an isotherm describes the partitioning of a
solute between the solid and liquid phases at equilibrium. Ion exchange resin Graver Technologies
*(1/n) C* S = K F CL

www.gravertech.com

! $ mass solute * CS = equilibrium conc. of solute on adsorbent # " mass dry adsorbent & % C L = equilibrium conc. of solute in fluid
*

! mass solute $ " volume of fluid %

K F = equilibrium constant (units depent on exponent) n = a constant
David R. Shonnard Michigan Technological University 70

3. Product Purification /Contaminant Removal: Adsorption Equilibrium- Freundlich Isotherm
Freundlich Isotherm:

0 < n <1
Favorable Adsorption

n =1 n >1

C* S

Unfavorable Adsorption

C* L
David R. Shonnard Michigan Technological University 71

3. Product Purification /Contaminant Removal: Adsorption Equilibrium- Langmuir Isotherm
Langmuir Isotherm:
* C* S,max C L C = * K L + CL * S

CS,max

*

C* S

Maximum Adsorption Capacity

C* L
David R. Shonnard Michigan Technological University 72

3. Product Purification /Contaminant Removal: Adsorption Equilibrium - Adsorbent Capacity
Example Problem:
Calculate the capacity of ion exchange resin to adsorb protein given that:

• m = mass of dry resin in a column = 1 kg • ε = porosity of the fixed-bed = 0.40 cm3 fluid/cm3 bed volume • ρr = resin density = 1.2 g dry resin/cm3 resin • n = 1 in the Freundlich Isotherm • per unit bed volume, there is 100 times more protein adsorbed as there is in the fluid at equilibrium. C*L = 1 mg protein/cm3 fluid at equilibrium

David R. Shonnard

Michigan Technological University

73

3. Product Purification /Contaminant Removal: Adsorption Equilibrium - Adsorbent Capacity
Problem Solution:
1. First, calculate KF in the Freundlich Isotherm.
* * is the Freundlich isotherm for n = 1 CS = K F CL

Basis of 1 cm 3 bed volume ' perform a solute mass balance " mass absorbed to resin in 1 cm 3 bed volume = 100 times the mass of protein in the fluid in the 1 cm 3 bed volume"
* * ρ r (1 − ε )(1 cm 3 ) = 100 C L ε CS * ε 100C L * or C = = K F CL ρ r (1 − ε ) * S

' KF =

100ε ρ r (1 − ε )

cm 3 fluid 100(0.4) KF = = 55.6 (1.2)(1 − 0.4) g dry resin
David R. Shonnard Michigan Technological University 74

3. Product Purification /Contaminant Removal: Adsorption Equilibrium - Adsorbent Capacity
Problem Solution:
2. Use the Freundlich Isotherm plus m= 1 kg resin to calculate capacity.

* Capacity = m C* S = m K F CL

! cm 3 fluid $ ! mg protein $ = (1,000 g dry resin)# 55.6 &"1 3 " cm fluid % g dry resin % = 55,555.6 mg Protein
For each kg dry resin

David R. Shonnard

Michigan Technological University

75

3. Product Purification /Contaminant Removal: Adsorption Equilibrium - Batch Adsorption
Example Problem 2: Batch Adsorption
An aqueous solution of protein (10 mg/cm3) of volume 1000 cm3 is contacted with 10 g of the resin (from the prior example problem). What is the concentration remaining in the aqueous phase after equilibrium is achieved? • m = mass of dry resin in a column = 10 g • n = 1 in the Freundlich Isotherm • V = volume of aqueous solution = 1,000 cm3. KF = 55.6 cm3 solution/g dry resin

Mass Balance on Protein
* C* S m + C L V = (10

mg protein )V 3 cm
76

David R. Shonnard

Michigan Technological University

3. Product Purification /Contaminant Removal: Adsorption Equilibrium - Batch Adsorption
Example Problem 2: Batch Adsorption (cont.)

Equilibrium C* = KF C*(1/n) = K F C* for n = 1 S L L Mass Balance Equation becomes: K F C L (100 g resin) + C L (1,000 cm ) = 10 mg Protein
3 4 C* ((100 g resin) K + 1,000 cm ) = 10 mg Protein L F * * 3 4

10 4 mg Protein C = cm3 fluid 3 ) + 1,000 cm ) ((100 g resin) (55.6 g resin
* L 3 = 1.52 mg Protein / cm C* L

David R. Shonnard

Michigan Technological University

77

3. Product Purification /Contaminant Removal: Adsorption Equilibrium - Batch Adsorption
Example Problem 2: Batch Adsorption (cont.)

* ! CL $ % Recovery of Protein = # 1 & 100 CLo % "

1.52 $ ! 100 = 84.76% = 1 " 10 %

David R. Shonnard

Michigan Technological University

78

3. Product Purification /Contaminant Removal: Fixed Bed Adsorption
Theory of Solute Movement in Fixed-Bed Adsorption Columns: (Blanch and Clark, “Biochemical Engineering”, pg 514-517)

ε = column viod fraction
(between particles) = VL / (VL + VS )

dz

β = particle void fraction
(voids within particles)

z

F uS = superficial velocity = ! $ " A% where F = volumetric flow rate A = cross sectional area of column
Michigan Technological University 79

0
David R. Shonnard

3. Product Purification /Contaminant Removal: Fixed Bed Adsorption
Theory of Solute Movement in Fixed-Bed Adsorption Columns: (cont.) 2 ∂(VCL ) ∂ (VCL ) ∂(VLC L ) ∂(VS s ) + + uS = DL ∂t ∂t ∂z ∂z2 (accumulation (accumulation (convective (axial

in liquid)

in solid) C Li (t, r,z)4π r2 dr 4 2 πR 3

flow)

dispersion)

s (t, z) = CLi β + ρ P CS CLi =

- - - avg. concentration inside particle 3 R 2 = r CLi (t, r, z)dr 3 (0 R DL = axial dispersion coefficient (cm2 / s)
Michigan Technological University 80

(

R

0

3 R 2 CS = r CSi (t, r,z)dr R 3 (0
David R. Shonnard

3. Product Purification /Contaminant Removal: Fixed Bed Adsorption
Theory of Solute Movement in Fixed-Bed Adsorption Columns: (cont.) Assumptions:

CSi >> CLi so s ≅ ρ P CS Neglect Dispersion, DL ≅ 0 ∂C L ∂C L ! 1 − ε $ ∂CS + ui + ρP = 0 " % ε ∂t ∂z ∂t Another Assumption: instantaneous equilibrium, CSi is uniform in the particles CS = CS = f(CL ) ! ∂CS $ ! ∂CL $ ∂CS ∂ CS ! ∂CL $ = f '(C = = # ) so L " ∂t % " ∂C L & % " ∂t % ∂t ∂t
David R. Shonnard Michigan Technological University 81

3. Product Purification /Contaminant Removal: Fixed Bed Adsorption
Theory of Solute Movement in Fixed-Bed Adsorption Columns: (cont.) Therefore

∂C L ui ∂CL + = 0 1ε ) ∂t ! $ f '(C ), ∂z 1+ ρ P" L . + ε % * This is the form of a kinematic wave. t2 t3 t1 Dispersion effects
CL

z
David R. Shonnard Michigan Technological University 82

3. Product Purification /Contaminant Removal: Fixed Bed Adsorption
Theory of Solute Movement in Fixed-Bed Adsorption Columns: (cont.) The velocity of solute propaga

! ∂C L $ " ∂t % dz ui − = − = dt ! ∂C L $ ) , !1-ε $ 1+ ρ ) f (C ' P" L . + " ∂z % ε % * the mean retention time of solute t= L) , !1-ε $f (C 1+ ρ ) ' P" L . ε % ui + * -

David R. Shonnard

Michigan Technological University

83

3. Product Purification /Contaminant Removal: Basics of Chromatography
• a solution containing a mixture of solutes (in a small volume) is added to the top of the column.
“Bioprocess Engineering: Basic Concepts” Shuler and Kargi, Prentice Hall, 2002

• a solvent (volume ∆V) is added to the top of the column.

• the solvent flow carries the solutes toward the bottom of the column.

David R. Shonnard

Michigan Technological University

84

3. Product Purification /Contaminant Removal: Basics of Chromatography
• each solute is carried along at a different apparent velocity, depending upon the strength of interaction with the column packing. • ideally, each solute exits the column as a discrete band of material.
“Bioprocess Engineering: Basic Concepts” Shuler and Kargi, Prentice Hall, 2002 time

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Michigan Technological University

85

3. Product Purification /Contaminant Removal: Basics of Chromatography
A technique to separate components in a mixture based upon differential affinity for solutes for the adsorbent. The affinity is quantified by the adsorption isotherm, CS* = f(CL*), and in particular the derivative, f ‘(CL*). The affinity could also include size selection as in gel permeation or molecular sieve chromatography.

David R. Shonnard

Michigan Technological University

86

3. Product Purification /Contaminant Removal: Theory of Chromatography
A Theory of Solute Movement
How much solvent (∆V) is needed to move a solute a distance ∆x? Solute balance over a differential column height ∆x

)! ∂CL $ , ! ∂CS' $ ! ∂CL $ ∆x . ∆V = ε A ∆x ∆V + A ∆x # ∆V -+ & % % " " " ∂V % ∂V * ∂x rate of solute rate of solute rate of solute removal by solvent flow removal from void space removal from solid phase

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Michigan Technological University

87

3. Product Purification /Contaminant Removal: Theory of Chromatography (cont.)

Simplifying Yields: ! ∂C L ∂CS' $ ∂C L + A #ε + & = 0 ∂x ∂V % " ∂V linear adsorption isotherm: CS = M f (CL ) amount of adsorbed solute per unit volume of column a function of CL mass of adsorbent per unit volume of column
'

David R. Shonnard

Michigan Technological University

88

3. Product Purification /Contaminant Removal: Theory of Chromatography (cont.)
Simplifying Yields: ∂ CL ∂C L = A (ε + M f '(C L )) − ∂x ∂V rearranging ! ∂V $ = A (ε + M f '(CL )) " ∂x % Integrating from xo to x and Vo to V ∆V ∆x = A (ε + M f '(C L )) distance that solute band moves
David R. Shonnard Michigan Technological University 89

elution volume of solvent

3. Product Purification /Contaminant Removal: Theory of Chromatography (cont.)
• The stronger the adsorption interaction, the shorter the travel distance, ∆x, for a given elution volume, ∆V.

adsorption isotherm

• a stronger adsorption interactions means a greater value of M f ‘(CL).

C'S
M f ' (CL )

CL

David R. Shonnard

Michigan Technological University

90

3. Product Purification /Contaminant Removal: Theory of Chromatography (cont.)
Example: Solute A, Adsorbent B
Adsorption isotherm: CS = k1 (CL)3

) mg A , + * mL solution . -

) mg A adsorbed , + . mg B * -

) mg A adsorbed , ) ! mg A $ 3 , . + . / +" % mg B mL solution * - * -

k1 = 0.2 CL = 0.05 mg A/mL solution ε = 0.35 M = 5 g adsorbent B/100 mL column volume ∆V = volume of solvent added = 250 mL [cm3] A = column cross-sectional area = 10 cm2

David R. Shonnard

Michigan Technological University

91

3. Product Purification /Contaminant Removal: Theory of Chromatography (cont.)
Find ∆x

f (CL ) = k1 C3 L f ' (C L ) = 3k1 C2 L

therefore

) ! mg A ads.$ , +# &. " %. mg B + = (3)(2)(0.05) = .0015 + ! mg A $ . + " mL soln.% . + . * M = 50 mg B 5gB = mL column volume 100 mL column volume

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Michigan Technological University

92

3. Product Purification /Contaminant Removal: Theory of Chromatography (cont.)
Find ∆x

∆x = =

∆V A (ε + M f '(C L ) ) ! 1 cm3 soln. $ 250 mL # & " mL soln. %

3 ) ! ! cm mg B mg A / mg B $ $ , soln. 2 + 50 3 +(10 cm )# 0.35 3 &&. # .0015 3 cm coln vol " mg A / cm soln.% % . cm coln vol " + * -

∆ x = 58.5 cm

David R. Shonnard

Michigan Technological University

93

3. Product Purification /Contaminant Removal: Examples of Chromatography
Bailey and Ollis, 1986, Fig. 11.18

David R. Shonnard

Michigan Technological University

94

3. Product Purification /Contaminant Removal: Examples of Chromatography
Bailey and Ollis, 1986, Fig. 11.19a, cation exchange chromatography

David R. Shonnard

Michigan Technological University

95

3. Product Purification /Contaminant Removal: Examples of Chromatography
Bailey and Ollis, 1986, Fig. 11.19b, cation exchange chromatography

David R. Shonnard

Michigan Technological University

96

3. Product Purification /Contaminant Removal: Examples of Chromatography
Gel Permeation Chromatography: is based on the penetration of
solute molecules into small pores of packing particles on the basis of molecular size and the shape of the solute molecules. It is also known as size exclusion chromatography.

Equivalent Equilibrium Constant K av,i = exp(-π L(rg + ri )2 )

where L = concentration of gel fiber (cm / cm 3 ) rg = radius of a gel fiber (cm) ri = radius of a spherical molecule of species, i (cm) K av,i is equivalent to f ' (CL ) in calculating t or
David R. Shonnard Michigan Technological University

dz dt
97

3. Product Purification /Contaminant Removal: Examples of Chromatography
Molecule radii estimated based on protein diffusion coefficients

Bailey and Ollis, 1986,

David R. Shonnard

Michigan Technological University

98

3. Product Purification /Contaminant Removal: Examples of Chromatography
Bailey and Ollis, 1986, Fig. 11.21, gel permeation chromatography

David R. Shonnard

Michigan Technological University

99

3. Product Purification /Contaminant Removal: Examples of Chromatography
Bailey and Ollis, 1986, Fig. 11.22, molecular sieve chromatography

David R. Shonnard

Michigan Technological University

100

3. Product Purification /Contaminant Removal: Ion Exchange of Amino Acids
Charged Amino Acid R Groups at Neutral pH

“Principles of Biochemistry” Lehninger, Worth, 1982

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Michigan Technological University

101

3. Product Purification /Contaminant Removal: Ion Exchange of Amino Acids (cont.)
Polar Amino Acid R Groups at Neutral pH

“Principles of Biochemistry” Lehninger, Worth, 1982

David R. Shonnard

Michigan Technological University

102

3. Product Purification /Contaminant Removal: Ion Exchange of Amino Acids (cont.)
Nonpolar Amino Acid R Groups at Neutral pH
“Principles of Biochemistry” Lehninger, Worth, 1982

David R. Shonnard

Michigan Technological University

103

3. Product Purification /Contaminant Removal: Ion Exchange of Amino Acids (cont.)
Acid dissociation reactions - Nonpolar and Polar R Groups:

“Bioseparaion Process Science” Garcia et al., Blackwell Science, 1999

David R. Shonnard

Michigan Technological University

104

3. Product Purification /Contaminant Removal: Ion Exchange of Amino Acids (cont.)
Acid dissociation reactions - Nonpolar and Polar R Groups:

“Bioseparaion Process Science” Garcia et al., Blackwell Science, 1999

David R. Shonnard

Michigan Technological University

105

3. Product Purification /Contaminant Removal: Ion Exchange of Amino Acids (cont.)
Acid dissociation reactions - Nonpolar and Polar R Groups:

“Bioseparaion Process Science” Garcia et al., Blackwell Science, 1999

David R. Shonnard

Michigan Technological University

106

3. Product Purification /Contaminant Removal: Ion Exchange of Amino Acids (cont.)
Acid dissociation reactions - Negatively Charged R Groups:

“Bioseparaion Process Science” Garcia et al., Blackwell Science, 1999

David R. Shonnard

Michigan Technological University

107

3. Product Purification /Contaminant Removal: Ion Exchange of Amino Acids (cont.)
Acid dissociation reactions - Negatively Charged R Groups:

“Bioseparaion Process Science” Garcia et al., Blackwell Science, 1999

David R. Shonnard

Michigan Technological University

108

3. Product Purification /Contaminant Removal: Ion Exchange of Amino Acids (cont.)
Acid dissociation reactions - Positively Charged R Groups:

“Bioseparaion Process Science” Garcia et al., Blackwell Science, 1999

David R. Shonnard

Michigan Technological University

109

3. Product Purification /Contaminant Removal: Ion Exchange of Amino Acids (cont.)
Acid dissociation reactions - Positively Charged R Groups:

“Bioseparaion Process Science” Garcia et al., Blackwell Science, 1999

David R. Shonnard

Michigan Technological University

110

3. Product Purification /Contaminant Removal: Ion Exchange of Amino Acids (cont.)
Acid dissociation reactions - Stoichiometry of COOH = HA:
[H+ ][A ] = [HA]

HA ← →
Ka

A+ H
+

-

+

'

Ka

[A-] log a K = log [H ] + log [HA]
+ pH = -log [H ]

and pK a = -log a K
[A-] (pH -pK a ) or = 10 [HA] or
= [HA] [A ] o (pH -pK )

[A-] pH = pK a + log [HA]
but [HA] = [HA] + [A ] o -

[A ] (pHpK a ) = 10 ] [HA] o - [A
David R. Shonnard

a [A ] 10 and = (pH -pK a ) [HA] 1 + 10 o

Michigan Technological University

111

3. Product Purification /Contaminant Removal: Ion Exchange of Amino Acids (cont.)
Acid dissociation reactions - Stoichiometry of NH3+ = HA+ :

HA

+

← →
Ka

A+ H

+

'

Ka

[H+ ][A] = [HA + ]

[A] + log a K = log [H ] + log + [HA ]
+ pH = -log [H ]

and pK a = -log a K
[A] (pH -pK a) or + = 10 [HA ] or = [HA [A] ] o - [HA ] [HA + ] 1 and + = (pH -pK a ) [HA ] 1 + 10 o
112
+ +

[A] pH = pK + log a [HA + ] but [HA ] o = [HA ] + [A]
+ [HA + ] [HA ] (pHpK a ) o = 10 + [HA ] + +

David R. Shonnard

Michigan Technological University

3. Product Purification /Contaminant Removal: Ion Exchange of Amino Acids (cont.)
Charge on amino acid groups:

Charge of α - amino group =

1 + 10 ( pH − pK )
c a

1(+ 1)

Charge of α - carboxyl group =

1+

Charge of side chain = 1+ Charge of side chain =
David R. Shonnard

10 ( pH − pK )
s a

1(− 1) 1

10 ( pH − pK )
a a

1(− 1) 1

1 + 10 ( pH − pK )
s a

1(+ 1)

Michigan Technological University

113

3. Product Purification /Contaminant Removal: Ion Exchange of Amino Acids (cont.)
Net Charge is sum of all reactions:
Net charge of amino acid = + c ( ) pH − pK a 1 + 10 1(+ 1) 1(− 1) 1

1+

Net charge of amino acid with (-R) =

+ c ( ) pH − pK a 1 + 10 1(+ 1)

1(+ 1)

10 ( pH − pK a )
a

1+

10 ( pH − pK )
a a

1(− 1) 1

+ 1+ +
a

Net charge of amino acid with (+ R) =

+ c ( ) pH − pK a 1 + 10

1+

10 ( pH − pK a )

1(− 1) 1

1 + 10 ( pH − pK a )
s

10 ( pH − pK a ) 1(+ 1)
s

1(− 1) 1

David R. Shonnard

Michigan Technological University

114

3. Product Purification /Contaminant Removal: Ion Exchange of Amino Acids (cont.)

David R. Shonnard

Michigan Technological University

115

3. Product Purification /Contaminant Removal: Ion Exchange of Proteins
A computer algorithm for computing charge of proteins (Genetics Computer Group, Inc. 1993)
) Net , )# of positively , )# of negatively , )# of protonated, )# of deprotonated , +ch arg e. = +charged residues. − +charged residues. + +amino termini . − +carboxy termini . * - * - * - * - * -

[H ] N ( p ) = N (t ) [H ] + K ( N )
+ +

N(p) is the number of protonated residues N(t) is the total number of residues of a specific type [H+] is the hydrogen ion concentration K(N) is the aminoacid dissociation constant
David R. Shonnard Michigan Technological University 116

3. Product Purification /Contaminant Removal: Ion Exchange of Proteins
Calculate the net charge of the following peptide NH2-Lys-Pro-Lys-COOH

Information Lysine positively charged
c = 2.19 pK a a pK a = 9.12 s pK a = 10.68

Information Proline nonpolar aminoacid
c = 1.9 pK a a = 10.41 pK a

) Net , )# of positively , )# of negatively , )# of protonated, )# of deprotonated , +ch arg e. = +charged residues. − +charged residues. + +amino termini . − +carboxy termini . * - * - * - * - * -

2
David R. Shonnard

0

1 from lys

1 from lys
117

Michigan Technological University

Information Lysine positively charged
c pK a = 2.19 a pK a = 9.12

Information Proline nonpolar aminoacid
c = 1 .9 pK a a = 10.41 pK a

pK as = 10.68

) Net , )# of positively , )# of negatively , )# of protonated, )# of deprotonated , +ch arg e. = +charged residues. − +charged residues. + +amino termini . − +carboxy termini . * - * - * - * - * -

2

) Net , H+ H+ H+ + + − + +charge. = 2 + −10.68 − 9.12 − 2.19 H H H + 10 + 10 + 10 * 3.5 3 Net charge 2.5 2 1.5 1 0.5 0 0 5 pH 10 15

[ ] [ ]

0

[ ] [ ]

1 from lys

[ ] [ ]

1 from lys

David R. Shonnard

Michigan Technological University

118

3. Product Purification /Nonideal effects on Chromatographic Separations
Dispersion, Wall Effects, and Nonequilibrium:

Gaussian Peak
σ i tmax,i = standard deviation

Resolution of Peaks tmax, j − t max,i RS = 1 / 2(t w ,i + tw, j )
“Bioprocess Engineering: Basic Concepts” Shuler and Kargi, Prentice Hall, 2002

David R. Shonnard

Michigan Technological University

119

3. Product Purification /Nonideal Effects on Chromatographic Separations
Prediction of Peak Width:

) ( t − t max,i )2 , yi = y max,i exp +2. + * 2(σ tmax,i ) . σ depends on dispersion and adsorption kinetics

σ2 =

v kal

v = superficial velocity, ka = surface adsorption reaction rate l = column length

David R. Shonnard

Michigan Technological University

120

3. Product Purification /Nonideal Effects on Chromatographic Separations
Prediction of Peak Height:

ymax,i is inversely proportional to σ t max,i
σ may depend on other processes

vd σ ∝ internal diffusion control, l v1/ 2 / d 3/ 2 2 σ ∝ external film control, l vd 2 2 σ ∝ Taylor dispersion (laminar flow), Dl
2

2

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Michigan Technological University

121

3. Product Purification /Scale Up of Chromatographic Separations
To Handle Increased Amount of Product: 1. Increase solute concentration using same column (may saturate column, leading to reduced purity) 2. Increase column cross sectional area, A, and particle diameter, d (maintains flow patterns, but σ increases if d increases) 3. Fix d but increase v and l, but maintain ratio of v to l constant (σ will be unchanged, but pressure drop will increase) 4. Increase A and volumetric flow rate, such that v is constant (σ remains constant, the desired outcome!)

David R. Shonnard

Michigan Technological University

122

3. Product Purification /Scale Up of Chromatographic Separations
Recent Advances in Chromatographic Packing:

1. Rigid beads with macropores inside particles 2. Allows higher flowrates without bead compression 3. Allows higher flowrates without excessive pressure drop 4. Good mass transfer is maintained between macropores and micropore within particles.

David R. Shonnard

Michigan Technological University

123

3. Chromatographic Separation of Proteins from Cheese Whey
Raw Milk Cream Separator Chandrasekaran, R., MS Thesis, Dept. of Chemical Engineering MTU

Heat Treatment Standardize (Optional) Cheese Milk Evaporator Rennet/Acid Spray Dryer NFDM Curds Cheese Ultrafiltration Evaporator/ Spray Dryer Whey

Ultrafiltration UF Milk

Acid Rennet/Heat

Whey Evaporator/ Spray Dryer MPC Evaporator/ Spray Dryer

Curds

Wash

Evaporator/ Spray Dryer Evaporator/ Spray Dryer Dry Whole Whey Casein

David R. Shonnard

WPCMichigan Technological University

Dry Whole Whey

124

3. Chromatographic Separation of Proteins from Cheese Whey (cont.)
Chandrasekaran, R., MS Thesis, Dept. of Chemical Engineering MTU

Table 1.2 Composition of Whey (Weight %) (Kosikowski et al., 1997)
Fluid Sweet Whey Water Total Solid Fat 93.7 6.35 0.5

Protein
Lactose Ash Lactic Acid

0.8
4.85 0.5 0.05

David R. Shonnard

Michigan Technological University

125

3. Chromatographic Separation of Proteins from Cheese Whey (cont.)
Whey proteins are finding increasing application in the fields of nutrition (protein powder), as an antibiotic, and in other pharmaceutical applications. Individual whey proteins can be separated using cation exchange chromatography, using pH change during elution to recover individual proteins.
Table 1. Isoelectric Points of Major Whey Proteins [1] Whey Protein Isoelectric Point 5.35-5.49 β-lactoglobulin α-lactalbumin Bovine Serum Albumin Immunoglobulins Lactoferrin Lactoperoxidase
David R. Shonnard

4.2-4.5 5.13 5.5-8.3 7.8-8.0 9.2-9.9
126

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3. Chromatographic Separation of Proteins from Cheese Whey (cont.)
Whey proteins have a range of molecular weights.

Table 2. Major Whey Protein Molecular Weights [1] Whey Protein Molecular Weight !-lactoglobulin 18,300 "-lactalbumin 14,000 Bovine Serum Albumin 69,000 Immunoglobulins 150,000 Lactoferrin 77,000 Lactoperoxidase 77,500

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Michigan Technological University

127

3. Chromatographic Separation of Proteins from Cheese Whey (cont.)

pH 6.5 to 11 step (Trial 1, 4/3/03)
0.9 0.8 0.7 0.6

20 mL HiPrep 16/10 SP XL
1

Flow Rate = 3 mL/min Temp. = 4 deg. C 300 mL of 5 g/L protein solution loaded pH 6.5
4

Absorbance

0.5 0.4 0.3 0.2 0.1

Elution
60 mL pH 6.5 buffer to 1 min. gradient to

3 2

0 0 20 40 60 80 100 120 140

60 mL pH 11 buffer

mL collected

David R. Shonnard

Michigan Technological University

128

3. Chromatographic Separation of Proteins from Cheese Whey (cont.)

Well 1 2 3 4 5 6 7 8 9

Sample MW Markers Lactoferrin Standard Peak 2a Colostrum pH 6.5 to 11 Peak 2b Colostrum pH 6.5 to 11 Peak 2c Colostrum pH 6.5 to 11 Peak 1 Trial 1 4/3/03 Peak 2 Trial 1 4/3/03 Peak 3 Trial 1 4/3/03 Peak 4 Trial 1 4/3/03

Lactoperoxidase and/or Lactoferrin appear to be in Lane 9, Peak 4.

David R. Shonnard

Michigan Technological University

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3. Effects of pH Gradient on Peak Resolution

500 ml of a solution of 5 g/L whey protein powder were loaded in the column HiPrep 16/10 SP XL and eluted using gradients from 0 to 85% pH11 (+ 15% pH 6.5 yielding pH 8.5 solution) in 2, 4, 6, 8, 10, 12 and 14 min, using program 2.
Program 2 Breakpoint Conc %B Flow rate Fraction Tube A Tube B (min) (ml/min) volume (ml) 0 0 3 5 pH 6.5 pH 11 20 0 3 5 pH 6.5 pH 11 (20+x) 85 3 5 pH 6.5 pH 11 (40+x) 85 3 5 pH 6.5 pH 11 (43+x) 100 3 5 pH 6.5 pH 11 (58+x) 100 3 5 pH 6.5 pH 11 Where x is the time for the pH gradient from 0% pH 11 to 85% pH 11.
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Valve position Load Load Load Load Load Load
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2 1 1

2

Figure 2. Change of 0% pH 11 to 85% pH 11 in 2 min.

Figure 3. Change of 0% pH 11 to 85% pH 11 in 4 min.

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Michigan Technological University

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2

1 1

2

3

Figure 4. Change of 0% pH 11 to 85% pH 11 in 6 min.

Figure 5. Change of 0% pH 11 to 85% pH 11 in 8 min

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Michigan Technological University

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3

3

2 1 1

2

Figure 6. Change of 0% pH 11 to 85% pH 11 in 10 min.

Figure 7. Change of 0% pH 11 to 85% pH 11 in 12 min.

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Michigan Technological University

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3

2 1

David R. Shonnard

Figure 8. Change of 0% pH 11 to 85% pH 11 in 14 min.

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4. Product Preparation / Crystallization
Crystallization is a nucleation process started from a concentrated solution: 1. Occurs when concentration exceeds saturation 2. Crystals have a well-defined morphology, large particle size 3. Homogeneous nucleation occurs when a solid interface is absent 4. Heterogeneous nucleation occurs when a foreign interface is present. 5. Secondary nucleation occurs in the presence of a crystal interface of the same solute
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4. Product Preparation / Crystallization
Critical cluster or nucleus is the largest cluster of molecules just prior to spontaneous nucleation: 1. n* is the number of molecules in the critical nucleus. 2. Subcritical clusters refers to when, n < n* 3. Supercritical clusters refers to when n > n* 4. An embryo is a cluster having n = n*. 5. An embryo or critical nucleus can range from 10 nm to several µm in size.

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Michigan Technological University

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4. Product Preparation / Crystallization
Steps in nucleation and crystal growth B + B ⇔ B2 + B ⇔ B3 + B ….. Bn-1 + B ⇔ Bn Bn + B ⇔ Bn+1
6

a critical cluster is formed which undergoes nucleation

Bn+1 + B ' which undergoes crystal growth

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Michigan Technological University

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4. Product Preparation / Crystallization
Characteristic zones of crystallization

New nuclei form spontaneously

Growth of existing crystals, Formation of new nuclei

No crystallization occurs

“Bioseparation Process Science” Garcia et al., Blackwell Science, 1999

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Michigan Technological University

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4. Product Preparation / Crystallization
Transport Processes During Crystallization

Growth Rate of Crystals, G = dL = kg ∆C dt where ∆C = C* − C is

Molecules in solution

the degree of saturation

Molecules in crystal
“Bioseparation Process Science” Garcia et al., Blackwell Science, 1999

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Michigan Technological University

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4. Product Preparation / Crystallization
Thermodynamics of Homogeneous Nucleation

Free Energy Change for Homogeneous Nucleation ∆ GHomogeneous = ∆ GSurface formation + ∆GClustering ∆ GSurface formation = 4π r 2γ sl C $ 4 / 3π r 3 ! = − RT ln * "C % V molar, solid
=
2 ! C $ 4 π rc = 0 = 8π rcγ sl − RT ln " * % C Vmolar, solid

“Bioseparation Process Science” Garcia et al., Blackwell Science, 1999, Pages127-140

where γ sl is the surface tension of the solid / liquid interface ∆ GClustering

The critical nucleus, rc , is where there is a maximum in ∆GHomogeneous d∆ GHomogeneous dr

2 γ sl Vmolar, solid rc = ! C$ RT ln " * % C
David R. Shonnard

Useful calculation when seeding a Crystallization process
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4. Product Preparation / Crystallization
Rate of Formation of Nuclei,dN/dt

Nucleation is analogous to reaction kinetics, ! ∆Gmax $ dN & B = = A exp# # & dt R T " %
0

! $ # & 3 2 # 16π γ sl & Vmolar, solid = A exp# 2 & ! C $$ & # 3R 3 T 3 ! # ln# * & & & # " " C %% % "
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4. Product Preparation / Crystallization
Batch Crystallization, Solid Phase Balance

1. Cummulative Number of Crystals, N versus size, L or 2. Population Density, n Slope of N vs L curve A balance on n tracks the number of crystals entering and leaving a specific size range due to crystal growth.
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4. Product Preparation / Crystallization
Batch Crystallization, Population Balance Equation

) Number of , ) Number of , ) Number of , ) Number of , +crystals initially . + +crystals growing. = +crystals at end . + +crystals growing. + . + . + . + . + * within range, L . - + *into range, L . - + * within range, L . - + *out of range, L . -

V ninitial ∆L + V G1 n1 ∆t = V nfinal ∆L + V G2 n2 ∆t V is volume, ∆L is size range, G is growth rate of crystal size (dL/dt), ∆t is a small time step. subscript 1 is a smaller size range, subscript 2 is size range for ∆L.
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4. Product Preparation / Crystallization
Batch Crystallization, Population Balance Equation (cont.)

divide by V , ∆L , and ∆t and allow ∆L and ∆t to go to 0. dn d (Gn) + =0 dt dL Assuming G is a constant over all L dn dn +G =0 dt dL
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Michigan Technological University

4. Product Preparation / Crystallization
Batch Crystallization, Population Balance Equation (cont.)

boundary conditions (BCs) for nucleation at t = 0, n = 0 B0 at L = 0, n = G as L → ∞, n is finite solve population balance equation and BCs using Laplace Transforms B0 ! Ls $ n= exp# − & in the Laplace Domain Gs " G% ! L$ n = B u# t − & " G%
0

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Michigan Technological University

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4. Product Preparation / Crystallization
Batch Crystallization, Cumulative Crystal Mass

M is cumulative crystal mass per unit volume M = ρ c k v ( n L3 dL
0 L

where ρ c is density of crystal solid and k v is a shape factor and as L → ∞, 1 M = W = ρ c kv B 0 G 3 t 4 4
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Michigan Technological University

4. Product Preparation / Crystallization
Batch Crystallization, Cooling Curve

Determine the time - temperature relationship to achieve a constant degree of supersaturation during batch crystallization rate of change of solute concentration = - rate of change of W dC dW =− dt dt dC = − ρ c kv B 0 G 3 t 3 dt
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4. Product Preparation / Crystallization
Batch Crystallization, Cooling Curve (cont.)

to achieve a constant degree of supersaturation, the rate of dC temperature change must be proportional to dt dC dT = kT = − ρ c kv B 0 G 3 t 3 dt dt integrating from the temperature that crystals start to form, T0 , at t = 0, we find that T0 -T =

ρ c kv B 0 G 3 t 4
4 kT
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4. Product Preparation / Crystallization
Continuous Crystallization, Solid Phase Balances

, ) Number of , ) Number of Number of , , ) + crystals growing . ) Number of +crystals growing. . . + + crystals leaving . + + crystals entering . = + + . . + out of range, ∆L , . + + into range, ∆ L, . + . + . + + * range, ∆ L, by flow. - + * range, ∆ L, by flow . + + * over a time, ∆t . *over a time, ∆ t .

V G1 n1 ∆t + Q nin ∆L ∆t = V G2 n2 ∆ t + V n∆ L ∆t V is volume, ∆L is size range, G is growth rate of crystal size ( dL / dt ), ∆t is a small time step, Q is volumetric flow rate through crystallizer. subscript 1 is a smaller size range, subscript 2 is size range for ∆ L , subscript in is for inlet conditions.
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4. Product Preparation / Crystallization
Continuous Crystallization, Solid Phase Balances

divide by ∆L , and ∆t and allow ∆ L and ∆t to go to 0, and assuming that no crystals are entering, nin = 0, and that G is constant. dn VG + Qn = 0 dL Restating in terms of residence time, τ = dn n + =0 dL G τ
o B Boundary Condition, L = 0, n = no = G

V Q

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Michigan Technological University

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4. Product Preparation / Crystallization
Continuous Crystallization, Solid Phase Balances

Population density solution, ! L $ n = n o exp# − & " Gτ% M = ρ c k v ( n L3 dL
0 L

where ρc is density of crystal solid and k v is a shape factor 1 1 3$ L $$ ! 3 3 ! 3 3 2 2 2 ! M = 6 ρc kv n G τ # G τ − " G τ + G τ L + G τL + L % exp" & % " 2 6 Gτ %
o

and as L → ∞ , M = W = 6 ρc kv n o G 4 τ 4
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4. Product Preparation / Crystallization
Continuous Crystallization, Advantages Advantages: 1. Input of solute helps to maintain a constant degree of saturation, ∆C 2. Desirable for determining growth rates and other kinetic parameters, but are not popular in industrial applications.

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Michigan Technological University

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