Membrane Technology for Enzyme Separations - NBV

Membrane Technology for Enzyme Separations
DSM
Membrane Technology
for Enzyme Separations
A case on efficient downstream processing
in enzymatic routes to cefalosporins
Emile van de Sandt and Marijn Rijkers

Membrane Technology for Enzyme Separations
Contents
• Introduction
DSM
– DSM
– The process
• The problem
• First results
• Process options
• Process design
• Project realization
• Outlook

DSM: Company Profile 2009
Membrane Technology for Enzyme Separations
Life Sciences & Materials Sciences Company
Global top 30 Chemical Industry
DSM
Net sales 2008: €9.3 billion
Net profit 2008: €577 million
23,591 employees
of which in R&D: approx. 2,200
of which in the Netherlands: approx. 7,200
>200 locations on 5 continents
No 1 or 2 in Dow Jones Sustainability Index
in 2004 to 2008
Strong technological toolbox:
Integrated use of biotechnology, biocatalysis,
organic chemistry, chemical and polymer
technology, material sciences

DSM: Ability to change
100 years of successful transformations
Membrane Technology for Enzyme Separations
Evolution
DSM
Coal
Fertilizers
Petrochemicals
Life Science Products
Performance Materials
1902 1930 1950 1970 1990 2000 2010
Classical Biotechnology
Bioterials / Biologics
sustainability
Technological competences
Mechanical engineering
Chemical engineering
Polymer technology
Material science
Fine chemicals
Modern Biotechnology

Membrane Technology for Enzyme Separations
DSM Antibiotics Supply Chain
DSM
Glucose
Side chain
Glucose
Side chain
Glucose
enzymatic
conversions
fermentation
Raw materials
Pen G 6-APA
7-ADCA
Intermediates
Amoxicillin
Ampicillin
(di)Cloxacillin
Flucloxallin
Cephalexin
Cefadroxil
Cephradine
Clavulanic acid
Nystatine
Enzyms
Bulk-actives
Penicillins
Cephalosporins
Other
Formulation Retail

Membrane Technology for Enzyme Separations
Innovation: Non-Natural Compound
Metabolic Engineering, Biocatalysis, Bioprocessing
DSM
13 chemical steps
replaced by: by
1 fermentation +
2 enzymatic steps
65% less energy and
50% lower cost
Life Cycle Analysis
Area use
Resource
consumption
and materials
Energy
consumption
1,00
0,50
0,00
Risk
potential
Emissions
Toxicity
potential

Membrane Technology for Enzyme Separations
DSM
Cefalexin Green Alternative (CEGA)
O
H NH 2
Enzymatic production process for Cefalexin
Start-up Start up: : 1997 in Barcelona
NH 2
+
H 2N
O
H H
N
S
CO 2
Pen-G-acylase
H 2 O
O
H NH2
Phenylglycine amide 7-ADCA ADCA Cefalexin
H
N
O
H H
N
S
CO 2

Membrane Technology for Enzyme Separations
Conversion
(Immobilized
enzyme)
DSM
Starting
Materials
Enzymatic Cefalexin Process Blockscheme
Solution
Product
isolation Recovery
Product Recycle
materials
Liquid
Purge

Membrane Technology for Enzyme Separations
DSM
Specification
R&D
Design / Construction
engineering
contruction
Start-up
FDA-approval
Pre-production
Market Testing
Project planning (beginning 1996)
1996
1997

Membrane Technology for Enzyme Separations
DSM
Description of problem
Product out of specification.
Dissolution test: Visual observation of turbidity
Poor control of protein content in reactor effluent
NOTE:
TM
Assemblase is still “in development”
Fast and robust solution required
Minimum interference with project targets and plans
Characterize proteins: Mw = 10.000 to 100.000
Develop technical solution: Choose Ultra Filtration

Membrane Technology for Enzyme Separations
Conversion
(Immobilized
enzyme)
DSM
Starting
Materials
Enzymatic Cefalexin Process Blockscheme
UF
Unit
Waste
Concentrate
Product
isolation Recovery
Product Recycle
materials
Liquid
Purge

Membrane Technology for Enzyme Separations
DSM
Ultra Filtration
Starting points
Full batch operation, batch size 4000 liter
Temperature 5°C
Retain Molecules with Mw > 20.000
Requirements
Losses to sewage < 0,3 % of feed
Effluent to recovery < 1,0 % of feed
Extra residence time < 75 minutes
Available washing liquid = 400 liter (minimize dilution)
Time available for washing / cleaning: 90 minutes
Water consumption for washing / cleaning < 1500 liter/batch
Construction
Sanitary design (no dead volumes, no cross-contamination, etc.)
Installation space 8x9x2,5 m (skid preferred)
Noise level: < 75 dB
Control
Normal operation is with local PLC control

Membrane Technology for Enzyme Separations
DSM
First experiments
1
2
3
Feed: Effluent of reactor containing (immobilized) enzyme
Equipment: Tubular membrane, area about 0,01 m 2 , cut-off 9 kD,
PolyEtherSulphon membrane (PCI). Cross flow velocity: 0.25 m/s.
Conditions: 3 bar, 5 °C. Concentration factor about 2.
Repeat with same feed.
Same equipment.
Conditions: 6 bar, 5 °C. Concentration factor about 4.
Repeat with feed containing (much) more proteins (worst case).
Tubular membrane, area about 0,01 m 2 , cut-off 8 kD,
PolySulphone membrane (PCI). Cross flow velocity: 0,25 m/s.
Conditions: 6 bar, 5 °C. Concentration factor between 4 and 5.

Membrane Technology for Enzyme Separations
DSM
Results
Product on spec. !
Flux = const * Δ P
High protein: fouling of membrane
Low crossflow and
fouling → lower flux
20 liter/m 2 /h attainable (lab.)
2. ES209, 6 bar, 5°C, CF=4
1. ES209, 3 bar, 5°C, CF=2 3. PU608, 6 bar, 5°C, CF=4-5

Membrane Technology for Enzyme Separations
DSM
Process choices
Batch versus continuous (“feed and bleed”): Choose Batch because of:
Fit with (batch) process.
Possibility for frequent cleaning (low risk).
Constant flow versus constant pressure: Choose constant feed flow
because of simpler design (no control valves).

Membrane Technology for Enzyme Separations
DSM
Process choices (continued)
1 versus 2 hours operation time: Choose 1 hour operation time.
Criterion
Investment (area)
Yield (hold-up)
Installation space
Cleaning fluid
Energy consumption
Flexibility
1 hour
+ Dfl 80000
higher ?
+ 6 m 2
+ 50%
+ 3 kW
higher
2 hours
-
-
-
-
-
-

Membrane Technology for Enzyme Separations
DSM
Membrane types
A. Tubular, typical “specific area”is 65 m2/m3
Drainage is possible, washing is difficult
B. Spiral wound, typical “specific area”is 2000 m2/m3
Drainage and washing is possible
Lab test: Filtration over a 20 kD membrane:
Obtain a permeate from which final product was isolated
with a protein content below detection limit.
Performance in turbidity test was good.
Product met specification.
Pilot tests:
Tubular and spiral wound membranes:
On spec product obtained.
No technical problems.

Membrane Technology for Enzyme Separations
DSM

Membrane Technology for Enzyme Separations
DSM
Process choices (continued)
Tubular versus spiral wound membranes: Not clear !
Criterion
Hold-up / conc. fact.
Drainability
Wash with small vol.
Fouling
Investment
Variable cost
Experience
Tubular
Much more !
65 m 2 /m 3
Good
Difficult
Lowest chance
More expensive
Little more
Available within
DSM
Spiral wound
Compact
2000 m 2 /m 3
Good
Good
Risk
Less expensive
-
Not within DSM

Membrane Technology for Enzyme Separations
DSM
Pilot tests (part of 2 nd CEGA pre-production)
PCI
Tubular membranes, same type as in lab tests.
Area 15,6 m 2 : Same scale as pre-production.
Attained fluxes: About 20 liter/m 2 /h.
Achieved concentration factor: 8-14 (not enough).
Amafilter “alternative”
Spiral wound membranes.
Area 1,6 m 2 : Side stream. However: Suitable to process
concentrate stream for PCI-unit (worst case).
Attained fluxes: About 13 liter/m 2 /h.
Achieved concentration factor: Up to 65 (enough).

Membrane Technology for Enzyme Separations
DSM
Result of pilot test (Amafilter)

Membrane Technology for Enzyme Separations
DSM
Options for full scale unit
Always start with maximum membrane area.
Reduce the area for maximum concentration factor.
Max. time: 75’, Max. amount of washing water: 400 liter/batch.
Membrane area’s according to quotations.
Criterion
Membrane area (m 2 )
Washing water (liter)
Required flux (l/m 2 /h)
Loss to recovery (%)
Loss to sewage (%)
Tubular
(13 stages)
203
200
26
2,1
0,12
Spiral
(4 stages)
230
160
17
0,7
0,22
Spiral
(3 stages)
288
200
16
0,6
0,28
Choose spiral wound membrane Ultra Filtration Unit

Membrane Technology for Enzyme Separations
DSM
Process design
A. Size of housings: Choose standard size of 4”, 4 meters long
B. Number of housings: 4
C. Columns and pumps must be “drainable”: install on slope
D. Pump requirements:
= sufficient cross flow velocity
= minimum heat input
= different speeds (4 speeds + reverse (for water intake))
= accurate (prefer positive displacement)
= sanitary design
Choose screw pumps type Mono
E. Heat exchange:
Requirements: Max. process temperature: 10 °C. Cool
to 5°C in 15 minutes. Heating to 50 °C must be possible
Secondary heating circuit (safety)
Minimum volume (use booster pumps on utility side)
Choose plate heat exchangers

Membrane Technology for Enzyme Separations
DSM
Design results
Install four UF-units which can be operated individually
Operations: Filling, filtration, draining, washing, rinsing, cleaning
Prepare dedicated program to operate the UF-unit
Number of washing steps
To recovery (% from feed)
Losses in purge (%)
Calculated efficiencies
Target 1 2 3

Membrane Technology for Enzyme Separations
DSM
Construction
Factory Acceptance Test (FAT)
At workshop supplier
Is the hardware installed (Installation Qualification, IQ)
Does it work (with water) ? Test pumps, controls,
alarms, valves (Operation Qualification, OQ)
Site Acceptance Test (SAT)
In plant
Does the integrated unit meet the targets ?
Waterbatches + number of production batches
= Process guarantees (yield + capacity)
= Pressure tests
= Alarms
= Noise
= Protocols, training

Membrane Technology for Enzyme Separations
DSM

Membrane Technology for Enzyme Separations
DSM

Membrane Technology for Enzyme Separations
DSM

Membrane Technology for Enzyme Separations
DSM

Membrane Technology for Enzyme Separations
DSM

Membrane Technology for Enzyme Separations
DSM

Membrane Technology for Enzyme Separations
DSM
Specification
R&D
Design / Construction
engineering
contruction
Start-up
FDA-approval
Pre-production
Market Testing
Project realisation (1997)
1996
1997

Membrane Technology for Enzyme Separations
DSM
Contributors....
DAI R&D team (Geleen / Spain) (Will v.d. Tweel, Eric Roos c.s.)
Membrane specialists (Veerle Cauwenberg, Paul Vergossen)
Amafilter (Manufacturer; Jan Gosker, André Wortel, Klaas Doedens)
DAI Chemferm maintenance (Antoni Serra c.s.)
DAI Production (Jordi Savall c.s.)
CEGA project (& start-up) team (Kees v. Helden c.s.)
MdE (Detailed engineering; M. de Morales c.s.)
DAI services (Purchasing, engineering, acceptance tests...;
Ben Voorn, Frits Leemker, Jerry Esmeijer, Ben Jansen + others)

Membrane Technology for Enzyme Separations
AreaPlant
=
Area
DSM
Lab
Volume
Volume
Plant
Lab
Area
Investment a0
a1
* + =
Relevant scale-up formula’s
=
Volume
Flux * Time
α
β
* f ( imp.
time)
* f ( process)
* f ( manuf
Area
Investment
Area
.)
Variable cost
γ

Membrane Technology for Enzyme Separations
DSM
Good options:
Further scale-up
Install fifth unit (up to 25% more capacity)
Increase pressure and optimize circulation.
Install membranes with higher cut-off.

Membrane Technology for Enzyme Separations
DSM
Good options:
Further scale-up
Install fifth unit (up to 25% more capacity)
Increase pressure and optimize circulation.
Install membranes with higher cut-off.
Better option:
Make process continuous.

Membrane Technology for Enzyme Separations
DSM
Good options:
Further scale-up
Install fifth unit (up to 25% more capacity)
Increase pressure and optimize circulation.
Install membranes with higher cut-off.
Better option:
Make process continuous.
Best option:
No protein release anymore (skip UF)

Membrane Technology for Enzyme Separations
DSM
Thank you !