12 Downstream Processing

Downstream Processing
(Textbook Chapter 11 &

Supplemental Chapter 11)
Topics
Downstream processing needed to separate cell mass & other
byproducts from the water solvent
Major categories
 Separation of solids & liquids
• Filtration
• Centrifugation
 Drying of solids
• Dryness limited by the dryness of air & adhesion of water to solid
 Separation of soluble components
• Adsorption – chromatography as special case
Updated: November 27, 2017

2
John Jechura (jjechura@mines.edu)
Typical process to manufacture enzymes
Bioprocess Engineering Principles, 2nd ed

Pauline Doran, Elsevier Science & Technology
Copyright © 2012, Elsevier Inc. All rights Reserved.

3
Need for downstream processing
Fermentation products are formed in dilute solutions

 Remove water without harming the products formed
 Byproducts?
Biological products are labile (i.e., sensitive to temperature,
solvents, etc.)
Fermentation broths are susceptible to contamination

4
General processing schemes
1. Cell removal 2. Cell disruption & cell debris

 If the cells are the product then removal
little additional processing may  Required if product is within the
be required cells
 Removal of the cells may make • Cell debris must then be
the separation of liquid species removed
easier  Option
 Options.. • High pressure homogenization
• Filtration
• Microfiltration
• Centrifugation

General processing schemes (cont.)
3. Primary isolation 4. Product enrichment

 Remove components that are very  Separation of product for high
different from the product concentrations
• May not be selective but  Option…
overall help the efficiency of • Chromatography
other separation
 Options… 5. Final isolation
• Solvent extraction  Depends on product, phase, &
final purity
• Two-phase liquid extraction
 Options…
• Adsorption
• Ultrafiltration (liquid)
• Precipitation
• Crystallization followed by
• Ultrafiltration
centrifugation, filtration, and
drying (solids)

Generalized downstream processing schemes


7
Water Content & Drying

Drying
Remove relatively small amounts of residual water and/or solvent

 May be necessary to minimize chemical or physical degradation
Relative terms – “dry” may range form 0 to 20 wt% water
Energy intensive operation

9
Water Content of Air
Absolute humidity defined as the ratio of the mass of water in dry

air
mW mW
Humidity   wW 
mair mW  mair
Relative humidity is the actual amount of water compared to the
maximum amount at the same pressure & temperature
pW
Relative humidity  sat
 100% where pW  yW P
pW
May also discuss properties of “wet bulb” temperature, …

10
Water Content of Air
Example: What is the wet bulb

temperature for air @ 80oF &
60% relative humidity?
Answer: 70oF
Ref: GPSA Data Book, 13th ed.

11
Water Content of Solids


12
Mechanisms of Drying
Moisture transport in solids

 Molecular diffusion of liquid water
 Capillary flow of liquid water within porous solids
 Molecular diffusion of vapor evaporated within the solid
 Convective transport of vapor evaporated within the solid
When the drying rate is controlled
by the vaporization of water from
the surface then the heat transfer &
mass transfer effects are balanced
hs Ah  Ta  T 

Nc    kG a  X  Xa 

Hvap

13
Drying Time
Can define the rate of drying to the water content of the solids
dX m
N  ms where X  w
dt ms
ms dX
n A  
A dt
Over the period of constant rate of drying
X ms

Nc  ms  t   X0  X1 
t N c

14
Drying Example – Humidity of Air
Dry sodium benzyl penicillin to a

moisture content of 20 wt% (wet
basis). Use air at 25oC & 1 atm
pressure in a fluidized bed drier.
Assume equilibrium (open circles)
Target dry basis water content:
20 kg water
X0   0.25  25
80 100 kg dry mass
Required relative humidity – 65% (from chart)
Mole fraction water – water’s vapor pressure @ 25oC is 3.17 kPa
pw 0.65 P sat 0.65  3.17 
yw     0.020
P P 101.28
15
Drying Time Example
Want to dry 10 kg washed filter cake (wet basis) from 15% to 8%

water (both wet bases) under constant drying conditions
 Drying area 1.2 m²
 Air temperature 35oC & surface temperature of solids 28oC
 Heat transfer coefficient 25 J/m²·sec·oC
 Drying time?
From initial conditions:
mw ,0 mw ,0
  0.15  mw ,0  1.5 and ms  8.5
mw ,0  ms 100
1.5
 X0   0.176
8.5

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Drying Time Example (cont.)
Final conditions
mw ,1 0.08  8.5
 0.08  mw ,1   0.74
mw ,1  8.5 1 0.08
0.74
 X1   0.087
8.5
Rate of drying
h A  T  T   251.2  35  28  5 kg
N c  s h a   8.62  10
H vap 2435.4  103 sec
Drying time
8.5
5 
t  0.176  0.087   8770 sec  2.4 hr
8.62  10
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Freeze Drying
Wet solids are frozen then exposed to vacuum – water directly

sublimes from solid to vapor state
Steps
 Freezing
 Primary drying (sublimation)
 Secondary drying (desorption)


18
Cell Removal

Filtration
Solid particles removed from a fluid suspension by forcing the fluid

through a filter media which holds back the solid particles
Ease of filtration depends on the properties of the solid & fluid
 Crystalline incompressible solids fairly easy to filter, …
 Fermentation broths not so much
• Small particle size & gelatinous nature
May use filter aids to improve efficiency of
filtration


20
Filtration Equipment
Plate & frame filter press

 Batch operation
 Accumulates solids. Must be opened & cleaned
 https://www.youtube.com/watch?v=M4wBd1_CvNw
 https://www.youtube.com/watch?v=aOQPr7Eomek
Rotary drum filters
 Can be operated continuously
 Can be operated at pressure with controlled atmosphere
 https://www.youtube.com/watch?v=zSflxzE0nIo
Horizontal belt filters
 Continuous operation
 Typically pull a vacuum below the filter
 https://www.youtube.com/watch?v=6voXE1HxYsY

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Filtration Theory
Rate of filtration depends on the pressure drop across the cake and
the filter media
1 dVf P P P
  
A dt  m     cV    cV 
 f   solids 
 mR  f   f   Rm   f   f 
  A     A    A 
 Rm is the resistance due to the filter media & is typically negligible

compared to the resistance due to the cake
 Various correlations to relate  (the specific cake resistance) to the cake
properties
• Compressible cake Rigid particles
Kv a2 1  
    P  
s
 3 p

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Filtration Theory
Can integrate the reciprocal form…

1 dVf P dt  Vf   f Rm
  A   f c  
A dt   cVf   dVf  AP  P
f     Rm 
  A  
t Vf V
 Vf 
f
 f Rm
A  dt    f c   dVf   dVf
0 0  AP  0
 P
 f c  Vf2   f Rm
At    Vf
2  AP  P
Linearized form
t  f c  f Rm
 2 Vf 
Vf 2A  P  A  P 

23
Filtration Example
Can filter 30 mL broth from penicillin fermentation on 3 cm² filter in

4.5 min using 5 psi pressure drop. Neglect media resistance & use
s=0.5:
    P     P 
s 0.5
Lab conditions to “tune” filter equation

 f   P  c 2A2t  P  2  3   4.5 5
0.5 0.5 2 0.5
t psi0.5min
 Vf   f c    0.201
Vf 2A  P 
2
Vf
2
30 
2
cm2
Filter 500 L broth in 1 hour using 10 psi pressure drop

 f   P  c  f c 
0.5
t
 Vf  A  Vf  11500 cm2  1.15 m2
Vf 2A  P 
2
2t  P 
0.5

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Filtration Example (cont.)
What if the cake is much less compressible? Let’s resize with s=0.1:
    P     P 
s 0.1
Lab conditions to “tune” filter equation
 f   P  c 2A2t  P  2  3  4.5 5
0.5 0.1 2 0.9
t psi0.9min
 Vf   f c    0.383
Vf 2A  P 
2
V f
2
30 
2
cm2
Filter 500 L broth in 1 hour using 10 psi pressure drop

 f   P  c  f c 
0.1
t
 Vf  A  Vf  10000 cm2  1.0 m2
Vf 2A2  P  2t  P 
0.9
Compressible cake will require more surface area

25
Centrifugation
Increase the “gravitational” field

experienced by particles to
promote faster settling times
Stoke’s law:
 p  L  p  L
ug  d p2g  uC  d p22 r
18L 18L
Z is the ratio of the acceleration due Pauline Doran, Elsevier Science & Technology
to the centrifuge relative to the acceleration due to gravity

(the g-force), 2r/g
 Industrial centrifuges can achieve 16,000, small lab centrifuges up to
500,000

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Equipment – Tubular Bowl Centrifuge
http://slideplayer.es/slide/7225569/24/images/11/EQUIPOS+DE+CENTRIFUGACI%C3%93N.jpg

27
Equipment – Disc Stack Bowl
Centrifuge


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Equipment – Disc Stack Bowl Centrifuge
Can be run in batch or continuous

modes
 Batch mode – equipment stopped,
opening up, & solids removed
 Continuous – either side discharge or
movement of solids out the top
• Disadvantage – the solids must
remain wet to allow them to move
Video: Alfa Laval PX Disc Stack
Centrifuge
 https://www.youtube.com/watch?v=
ZmTMz4VkAwI http://www.alfalaval.com/globalassets/images/products/se
paration/centrifugal-separators/disc-stack-separators/sx-
bowl_440x360.png

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Equipment – Decanter Separator
Andritz 2-phase decanter with CIP (clean in place)

 Video: https://www.youtube.com/watch?v=OqEODWcJwnY
http://www.gn-decanter-centrifuge.com/mud/wp-content/uploads/2012/09/decanter-1.png

30
Centrifuge Performance
Relate the performance based Disc stack bowl centrifuge

on the centrifuge’s cross-
sectional area to that of a 22  N  1
gravity separator 
3g tan   
 3
r
2  r13 

Q

2ug Tubular bowl centrifuge
Two centrifuges that operate 2b 2 2 22br 2

with equal effectiveness

2g
 3r2  r1  
g
1 Q
Q 2

1 2

31
Centrifuge Example
Use continuous disc stack centrifuge @ 5000 rpm to recover 50% baker’s yeast
cells at 60 L/min.
 Found that at constant rpm the solids recovery factor is inversely proportional to flow rate
At 5000 rpm what flow rate will give 90% solids recovery?
1
Y  Q  1 Y1   60  0.5  33.3 L/min
2  Q

Q Y2 0.9
What rpm necessary to get 90% recovery @ 60 L/min?
 At equal effectiveness by adjusting flowrate:
1 Q
Q 2 2 Q 2 33.3
     0.556
1 2 
1 Q1 60
 By adjusting rotational speed:
2 22 1
 0.556  2  2   6710 rpm
1 1 0.556
32
Separation of Soluble
Components

Separation of Soluble Components
Liquid-liquid extraction
Precipitation
 Followed by solid/liquid separation & possibly drying
Adsorption
 Chromatography a special case
Membrane separations
 Dialysis (separation by selective membrane)
 Reverse Osmosis – transport of water from low to higher concentration
 Ultrafiltration & microfiltration
 Electrodialysis
Crystallization

34
Absorption vs Adsorption
Absorption Adsorption
From Prof. Art Kidnay

35
Adsorption
Chemical species cling to the surface of the adsorbent allowing

species that don’t adsorb to pass on by
Equilibrium relationship between components in liquid & to surface
CLSm KLCL*
C  KF  C 
1/ n
*
S
*
L (Freundlich) C 
*
S (Langmuir)
1 KLCL*
Differential material balance in a packed bed column
CL C C
u   L   1   S
z t t
Including overall mass transfer coefficient
dCS CL C
  KLa   CL  CL*   u   L   1   KLa   CL  CL* 
dt z t

36
Adsorption (cont.)
Approximate steady flow through fixed-bed adsorption column as a

steady-state moving-bed operation
dCL
u   1   KLa   CL  CL* 
dz
Can integrate to determine the height of a column for a particular
degree of separation
H CL 0
u dCL
H   dz  
0 1   KLa  CL
CL  CL*
Need the equilibrium relationship to fully integrate

37
Adsorption (cont.)
Mass balance considerations on the adsorbent

provides an operating line & relates the
concentrations in the liquid & on the solid
F  CL0  0   B  CS* 0  0 
B CL CL0
  *
F CS CS 0
F is the liquid volumetric flow (uA) & B is the

resin’s volumetric flow

38
Adsorption (cont.)
Fixed beds can be thought to have three zones:

 Saturated zone
 Adsorption zone – also called the mass transfer zone
 Virgin zone
We would stop flow before the adsorption zone gets to the exit
(i.e., before the virgin zone goes completely to zero)
If we let the zones travel
through the column then
we would get a typical
breakthrough curve

39
Adsorption (cont.)

40
Adsorption Example
Separate cephalosporin on to an ion-exchange resin

 Bed is 4 cm diameter
 Resin density is 1.3 g/cm³ & packed to 0.8 cm³ resin per cm³ bed
 Feed solution CL0 = 5 g/L
 Broth superficial velocity u = 1.5 m/h & broth to resin feed ratio F/B = 10
 Equilibrium relationship CS = 25 (C*L)1/2 & mass transfer KLa = 15 h-1
 What is the height necessary to get CL = 0.2 g/L?
B CL CL0 1
  *   CS  10 CL and CS* 0  10 CL0
F CS CS 0 10
1 *2 1
C  25  C  10 CL   0.16 CL2
1/2 2
*
S
*
L  CL*  CS 
625 625

41
Adsorption Example (cont.)
Substitute into integral equation for the height

CL 0
u dCL
1   KLa  C
H
L
CL  CL*
5
1.5 dCL
 0.815 0.2 CL  0.16 CL2

5
1.5   CL 
 ln  
    
0.8 15 1  0.16 CL   0.2
 0.6 m
Amount of resin needed?

  4
2
D 2
mresin  resin H  1.3   60   980 g
4 4
42
Chromatography
Form of adsorption where chemical species adsorb & desorb at

different rates
 Those species that interact most strongly with the stationary phase will elute
later than those species that weakly interact
 Separation of a “pulse” of mixture
• Followed up by more solvent
• Recovered material separated from the other species of the original
mixture but diluted with more solvent
• Can obtain multiple products from each pulse
We’ll focus on chromatography as a batch process
 Continuous & semi-continuous options
• Moving Bed Chromatography
• Simulated Moving Bed Chromatography
43
Chromatography
Concentrations in the separation

of mixture with 3 solutes
Species that adsorb onto the
stationary phase the least will
elute the earliest


44
Chromatography Methods
Adsorption chromatography (ADC) Affinity chromatography (AFC)

 Controlled by weak van der Waals  Based on specific chemical
forces & steric interactions interactions between solute molecules
& ligands
Liquid-liquid partition
 Possible to get very high resolution
chromatography (LLC) (degree of separation) but expensive
Ion-exchange chromatography (IEC) sorbent
 Adsorption of ions on resin particles Hydrophobic chromatography (HC)
by electrostatic forces
 Widely used, especially for protein High-pressure liquid chromatography
recovery (HPLC)
Gel Permeation Chromatography –
Size Exclusion Chromatography
 Smaller molecules wander into the
pores of the solid

45
Chromatography Separation of Peaks
The species will move through the chromatography column slower

than the rest of the solvent – differential migration
Given enough
column length the
species will elute
from column with
little to no overlap


46
Chromatography Zone Spreading
Elution bands spread out so each solute takes time to pass across
the end of the column
Factors that lead to zone spreading
 Axial diffusion
 Eddy diffusion
 Local nonequilibrium effects
Minimize zone spreading by improving mass transfer more closely
approach equilibrium between the phases
 Good: increasing the particle surface area per unit volume
 Bad: increasing liquid flow rate

47
Descriptors
 Ve – volume of eluting solvent needed to carry the solute through column
 Vo – void volume in the column (·Vempty)
• In gel chromatography (size exclusion) there is also a volume term for the
interstitial volume only accessible by the smaller molecules
 ki – capacity factor for species “i”
V  Vo
ki  e ,i
Vo
  – selectivity between components “1” & “2”
k
 1
k2

48
Descriptors based on solute peaks as normal distributions

 ymax,i – Maximum concentration of eluting peak
 tmax,i – Time at which peak elutes
  – Standard deviation of peak shape
  t  t 2 
y i  ymax, i exp   
max, i
 2  tmax,i  
2
 

49

 Yi –Yield of solute (ratio amount recovered
to total in original mixture & recoverable)
t
 Vy
 i dt
Yt ,i  0

 Vy
0
 i dt
 Cumulative yield for normal distribution
1    t  tmax,i   
Yt ,i  1 erf  
2
  2  tmax,i   
 Incremental yield the difference between the cumulative yields @ two times

50

 Excel has functions for normal Gaussian distributions – scaled so that the
cumulative amount is 1
Cumulative: NORM.DIST( x, mean, standard_dev, TRUE )
Differential: NORM.DIST( x, mean, standard_dev, FALSE )

51

 2 is essentially a Peclet number – ratio of flowrate to reaction rate
u V / A
 
2

ka L ka L
• Value depends on the rate controlling step

ud 2
 
2
Internal diffusion control
L
1/2 3/2
u / d
2  External film control
L
2
ud
2  Taylor diffusion
DL

52
Descriptors based on HETP concept

 N – Number of theoretical stages (theoretical plates)
2
V 
N  16  e 
w
 HETP – Height of a theoretical plate (L = length of column)
L
H
N


53
Resolution – ratio distance of solute to solvent

 Resolution between two adjacent peaks
tmax, j  tmax,i
RS 
1
2
 tw , j  tw , i 
tmax, j  tmax,i 2 Ve ,2  Ve ,1 
 
1
 4 j tmax, j    4i 4tmax,i   w2  w1
2 
 Assume the spreading is about the same
(w2 = w1) & use concept of number of theoretical
stages…
Ve ,2  Ve ,1 1 Ve ,2  Ve ,1 1    1  k2 
RS   N  N  
w 4 Ve ,2 4     k2  1

54
Chromatography Material Balance
Assume downward flow through a fixed-bed chromatography

column – adsorbed species come to very quick
equilibrium with the adsorbent
Following a small pulse (moving reference system)
CL  CL CS 
 A  0
x  V V 
Integrated form…
V
x 
A    M f  CL  
Uses equilibrium relationship…

CS  M f  CL  and f  CL   d f/ dCL

55
Chromatography Material Balance Example #1
Chromatography column 10 cm long with cross sectional. Packed with adsorbent,

=0.35 & M=50 mg adsorbent per mL column volume. Adsorption isotherm given
by:
f  CL   0.2 CL3
Determine position of the solute band & solvent front when ΔV=250 mL &
CL=0.05 mg/mL
 Solvent front:
V 250 cm3
x    71.4 cm
A 10 cm   0.35
2
 Solute:
f  CL   0.6 CL2 f  CL   0.6  0.05  0.0015
2

V 250
x    58.8 cm
A   M  f  CL   10 0.35  50  0.0015 
56
Ion-exchange chromatography to purify 20 g of Protein A.

Operate column @ 20 cm/hr. Peak exits at 80 min (tmax) with 10
min standard deviation ( tmax).
How long must the column be run to get 95% yield?
1    t  tmax,i   
Yt ,i  erf    1
2   2  tmax,i   
   
1    t  80   
 erf    1  0.95
2   2 10   
 
 By iterative solution t = 96.4 min

57
Let’s double the flowrate to 40 cm/hr. Assume the peak spreading

depends on Taylor diffusion.
How long must the column be run to get 95% yield?
 Adjust the tmax &  parameters
tmax,2 u1 20 cm/hr
   0.5  tmax,2  40 min
tmax,1 u2 40 cm/hr
22 u2  10 
 2  2  1 2    2  0.177
1 u1
2
 80 
 By iterative solution t = 51.6 min
1  
Yt , i  erf 
 t  40   
  1  0.95
2   2  0.177  40   
 
58
Material Balance Examples #2 & #3
Example #3 requires relatively more time because of the change in

peak shape at the two velocities

59
Summary

Summary
Downstream processing needed to separate cell mass & other

byproducts from the water solvent
Major categories
 Separation of solids & liquids
• Bulk separation – chemical species remain in their respective phases
• Types
o Filtration
o Centrifugation
 Drying of solids
• Dryness limited by the dryness of air & adhesion of water to solid
 Separation of soluble components
• Adsorption – chromatography as special case

61

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Downstream Processing

(Textbook Chapter 11 &

Updated: November 27, 2017

Bioprocess Engineering Principles, 2nd ed

Updated: November 27, 2017

Fermentation products are formed in dilute solutions

Updated: November 27, 2017

1. Cell removal 2. Cell disruption & cell debris

Updated: November 27, 2017

3. Primary isolation 4. Product enrichment

Updated: November 27, 2017

Bioprocess Engineering Principles, 2nd ed

Updated: November 27, 2017

Updated: November 27, 2017

Remove relatively small amounts of residual water and/or solvent

Updated: November 27, 2017

Absolute humidity defined as the ratio of the mass of water in dry

May also discuss properties of “wet bulb” temperature, …

Updated: November 27, 2017

Example: What is the wet bulb

Ref: GPSA Data Book, 13th ed.

Bioprocess Engineering Principles, 2nd ed

Updated: November 27, 2017

Moisture transport in solids

Updated: November 27, 2017

Over the period of constant rate of drying

Updated: November 27, 2017

Dry sodium benzyl penicillin to a

Want to dry 10 kg washed filter cake (wet basis) from 15% to 8%

Updated: November 27, 2017

Wet solids are frozen then exposed to vacuum – water directly

Bioprocess Engineering Principles, 2nd ed

Updated: November 27, 2017

Updated: November 27, 2017

Solid particles removed from a fluid suspension by forcing the fluid

Bioprocess Engineering Principles, 2nd ed

Updated: November 27, 2017

Plate & frame filter press

Updated: November 27, 2017

 Rm is the resistance due to the filter media & is typically negligible

Updated: November 27, 2017

Can integrate the reciprocal form…

Updated: November 27, 2017

Can filter 30 mL broth from penicillin fermentation on 3 cm² filter in

Lab conditions to “tune” filter equation

Filter 500 L broth in 1 hour using 10 psi pressure drop

Updated: November 27, 2017

Lab conditions to “tune” filter equation

Filter 500 L broth in 1 hour using 10 psi pressure drop

Compressible cake will require more surface area

Updated: November 27, 2017

Increase the “gravitational” field

to the centrifuge relative to the acceleration due to gravity

Updated: November 27, 2017

Updated: November 27, 2017

Bioprocess Engineering Principles, 2nd ed

Updated: November 27, 2017

Can be run in batch or continuous

Updated: November 27, 2017

Andritz 2-phase decanter with CIP (clean in place)

Updated: November 27, 2017

Relate the performance based Disc stack bowl centrifuge

Two centrifuges that operate 2b 2 2 22br 2