novel liquid-liquid scalable extraction/chromatography technology · 2016-10-05 · optimising...

Optimising Biomanufacturing

December 2-3, 2008Novel Liquid-liquid Extraction Technology

Novel liquid-liquid scalable extraction/chromatography technology

December 3rd, 2008

Professor I.A. Sutherland



Delft, Utrecht, Rotterdam, BPP2005

• Introduction to industrial scale centrifugal liquid-liquid extraction and its potential for the purification and manufacture of proteins and biologics

• Reviewed Hydrostatic & Hydrodynamic Countercurrent Chromatography

• Prospect for true moving bed chromatography



Outline of Today’s Talk

• Some advantages of working with liquid stationary phases

• Some additional not well known versatilities

• Quick review of high-resolution, hydrodynamic counter-current extraction

• Quick introduction to aqueous two-phase systems (ATPS)

• Focus on high-resolution, hydrostatic counter-current extraction using ATPS in isocratic mode

• Introduction to intermittent true moving bed extraction

• Other high resolution continuous extractions schemes in the offing



Key Advantages of a Liquid Stationary Phase

• Total (100%) sample recovery – liquid-liquid nature of the process means it is gentle and versatile and there is no irreversible adsorption to a solid support

• Improved sample solubility – both upper and lower phase available

• Quick due to higher throughput – High flow, low pressure system

• Inexpensive & a Green Technology compared to HPLC (significantly less solvent usage and no expensive solid supports)

• Crude extracts, including particulates can be handled reducing the number of separation steps in a process and reduced sample preparation requirements

• High loading capacity due to liquid nature of stationary phase

• Easy & predictable scale-up so that scale-down to scale-up philosophy can be applied

• Low pressure system – so columns can be lengthened to increase resolution and widened to increase capacity and throughput

• Normal and reversed-phase operation and the switching between them gives expanded operational capability – elution extrusion, dual mode, true moving bed



Other Little Known Advantages

• Isocratic operation– Either phase can be mobile (normal or reverse phase

– The process can be switched half way through a run

– The process can be stopped and the retained components pumped

out (elution/extrusion – Berthod)

• Co-current Chromatography/Extraction– Both phases can be pumped at the same time in the same direction

• Counter-current Chromatography/Extraction– Both phases are simultaneously flowed in opposite direction

– Continuous processing of closely related binary compounds



Hydrodynamic Counter-current Extraction



New Dynamic Extraction/Chromatography Technology

• Developed now as a fast, robust, continuous process with separations in minutes as opposed to hours with previous technology

• Small molecules – Natural Products

• Potential for application to proteins, peptides, plasmids and other biologics



Coil Planet Centrifugation producing mixing

and settling zones along a continuous tube



Twist free connection to rotor



DE Centrifuges - Predictable Scale-up

Milli-CCC – R=50mm, Vc=5-17ml Brunel-CCC – R=110mm, Vc=0.1-1 litre Maxi-CCC – R=300mm, Vc=4.6 litre

CCC-Device

Bore Area B Mean

Flow

92.5 90 85 80 70

Mini-DE 0.8 0.50 -37.01 0.50 0.04 0.07 0.16 0.29 0.66

Midi-DE1 1.6 2.01 -11.22 2.00 0.45 0.79 1.79 3.18 7.15

Midi-DE2 3.68 10.64 -3.13 10.00 5.74 10.21 22.97 40.83 91.87

Maxi-DE 10 78.54 -0.82 80.00 83.66 148.72 334.62 594.88 1338.49

Flow at SP Retention (Sf) given below



Hydrodynamic Counter-current Extraction

• Continuous tube with wave mixing

• Stop the pump & liquid phases move to opposite ends of the tubing

• Ideal with low viscosity aqueous organic phases

• Hydrodynamic equilibrium between the mobile phase and the retained liquid stationary phase

• Kg scale in days have been demonstrated with aqueous/organic phase systems

• Feasibility of continuous countercurrent extraction has now been demonstrated at a pilot scale



Aqueous Two-Phase Polymer Systems (ATPS)



Aqueous Two-Phase Systems (ATPS)•Numerous polymer-polymer, polymer/salt combinations

•Gentle host medium for biologics (ref Albertsson, Kula, Hubbuch)

•Low interfacial tension

•ATPS more viscous than aqueous/organic phase systems



Cascade v. Wave Mixing




Hydrodynamic systems retain ATPS

but wave mixing is too gentle for

large proteins




Hydrodynamic systems retain ATPS

but wave mixing is too gentle for

large proteins

Large scale toroidal hydrostatic

systems being constructed but

not yet tested



Hydrostatic Counter-current Extraction



Hydrostatic Counter-current Extraction

• Often discrete chambers with narrow bore interconnecting tubes

• Cascade mixing (like waterfalls)

• Stop the pump and the stationary phase stays where it is – trapped in the chambers

• The focus today - Centrifugal Partition Chromatography (CPC)

Channel

Duct

CPC: ... Without solid support ...

G

Descending modeFlow

Channel

DuctFlow

Ascending mode

PUMP

PUMP

1 channel ≈ 90 µL

Sanki HPCPC, analytical scale

Armen Elite, industrial scale

Twin cells ≈≈≈≈ 12 mL

For the 12.5 liter instrument

Earth gravity modulates the Coriolis acceleration and promotes spray formation

Earth gravity :possible sedimentation of the lower phase in deep channels

Vertical pack Horizontal pack

Visual CPC II

g

g gΓcor.

Γcor.

Stroboscopic looking at the same cell, observing

the spray formation

Stroboscopic flash and

fast response camera

1) Marchal L, Foucault AP, Patissier G,

Rosant J, Legrand J (2002) Centrifugal partition chromatography: an engineering

approach, In: Comprehensive Analytical

Chemistry (A Berthod, Ed), Elsevier, 115-

157.

2) Marchal, L., Legrand, J., Foucault, A. (2003)

Centrifugal partition chromatography: a

survey of its history and our recent

advances in the field, Chem. Rec. 3(3),

Flow rate (mL/min)

BuOH / Water

Ascending Mode

From 0 to 500 mL / min in 1 min then back to 0 mL / min in 1 min

02550751001251501752002252502753003253503754004254504755004754504254003753503253002752502252001751501251007550250

1290 rpm506 rpm



Advanced Bioprocessing Centre (ABC)

Hazard’s Lab

Applications Lab

Analytical Lab

Control Room



1 litre Hydrostatic CPC Set-up at BIB



Run Conditions

• Centrifuge: Armen Elite 1 litre CPC

• Capacity: One rotor only - volume 500ml, cell volume 429ml

• Phase System: ATPS: 12.5% PEG1000-12.5% K2HPO4

• Sample: 90mg lysozyme, 90mg myoglobin in 40ml (~10% CV) 50:50 ATPS mix

• Speed: 2000 rpm (224g)

• Flow: 10 ml/min

• Mobile Phase: lower salt phase

• Stationary phase retention (Sf) at breakthrough: 52%

• Stationary phase retention (Sf) at end: 19%



Analytical Runs

0

10000000

20000000

30000000

40000000

50000000

60000000

70000000

80000000

90000000

100000000

0 10 20 30 40 50 60 70 80

Fraction Number

HP

LC

Peak

Are

a (

uV

s)

Lysosyme

Myoglobin & Apomyoglobin

k=0.59, Sf=40%, 80 CCD steps

k=1.91, Sf=19.5%, 80 CCD steps

Rs=1.28



Analytical Runs

0

10000000

20000000

30000000

40000000

50000000

60000000

70000000

80000000

90000000

100000000

0 10 20 30 40 50 60 70 80

Fraction Number

HP

LC

Peak

Are

a (

uV

s)

Lysosyme


k=0.59, Sf=40%, 80 CCD steps

k=1.91, Sf=19.5%, 80 CCD steps

Note: theoretical prediction

from test tube experiments



Transfer to Pilot Scale Unit in France



Armen 12.5 litre CPC at Archimex



Run Conditions

• Centrifuge: Armen Continuum 12.5 litre CPC

• Capacity: One rotor only - volume 6.25 litre


• Sample: 1.1g lysozyme, 1.1g myoglobin in 500ml (~10% CV) 50:50 ATPS mix

• Speed: 1293 rpm (224g)

• Flow: 125 ml/min

• Mobile Phase: lower salt phase

• Stationary phase retention (Sf) at breakthrough: 63%

• Stationary phase retention (Sf) at end: 22%



Some of the team at Archimex



Fraction collection & on-line monitoring



Armen 12.5 litre CPC at Archimex

0

200

400

600

800

1000

1200

1400

1600

0 10 20 30 40 50 60 70 80

Fraction Number

HP

LC

Pe

ak A

rea

Lysozyme

Myoglobin + Apomyoglobin

Rs = 1.88, sample 10%CV

ECM172 - 10%CV Sample Loading

0

10000000

20000000

30000000

40000000

50000000

60000000

70000000

80000000

90000000

100000000

0 10 20 30 40 50 60 70 80

fraction Number

HP

LC

Pe

ak

Are

a

Lysosyme


Rs=1.28



Theoretical Predictions – Process Scale

0

200

400

600

800

1000

1200

1400

1600

0 10 20 30 40 50 60 70 80

Fraction Number

HP

LC

Pe

ak A

rea

Lysozyme

Myoglobin + Apomyoglobin

Theory - k=0.59, Sf=52.5%, 80 CCD steps

Theory - k=1.91, Sf=24.2%, 80 CCD steps



ATPS Stripping Characteristic – Scale-up

ATPS Stripping Characteristic During Scale-up

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 10 20 30 40 50 60 70 80 90

Time (mins)

Fra

cti

on

of

Co

lum

n V

olu

me

6.25 litre rotor

0.5 litre rotor



Conclusions – Batch Isocratic

• Linear scale-up feasible

• Robust technology – easily transferred from the test tube to a production run

• Note that with Hubbuch’s robotic high throughput screening with ATPS systems, combined with this rapid scale-up approach – small footprint, rapid new approaches to manufacture are becoming feasible



Intermittent – True Moving Bed (i-TMB)



True Moving Bed

T(P)

Heavy Phase Out

Light Phase In

Light Phase Out

Heavy Phase In

Force Field

(ng)

H(C)

Sample In

Distal – outer layer

Proximal – inner layer

Sutherland, Scientific Update, San Francisco

March 21-22, 2005



Injection

Ascending mode period

Descending mode period

Injection of A + B

BA



Run Conditions

• Centrifuge: Armen Elite 1 litre CPC

• Capacity: Two rotors - volume 1000ml, cell volume 858ml


• Sample: Continuous flow of 2.2mg/ml lysozyme, 2.2mg/ml myoglobin at 3ml/min in UP (ascending) and LP (descending) –0.79g/hr – total 1.2g in 1.5 hours

• Speed: 2000 rpm (224g)

• Flow: 10 ml/min (Ascending) 5 minutes; 10ml/min (Descending) 5 minutes and so on …..

• Mobile Phase: alternating between between UP & LP



Armen CPC i-TMB System at Archimex



Descending Elution

0

1000

2000

3000

4000

5000

6000

7000

8000

0 20 40 60 80 100 120 140 160

Fraction Number

HP

LC

Pe

ak

Are

a (

arb

. u

nit

s)

Lysozyme




Ascending Elution

0

1000

2000

3000

4000

5000

6000

0 20 40 60 80 100 120 140

Fraction Number

HP

LC

Pe

ak

Are

a (

arb

. u

nit

s)

Lysozyme

Myoglobin + ApoMyoglobin



D e s c e n d i n g E l u t i o n

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

6 0 0 0

7 0 0 0

8 0 0 0

0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0

F r a c t i o n N u m b e r

HP

LC

Pe

ak A

rea

(a

rb.

un

its)

L y s o z y m e

M y o g l o b i n & A p o m y o g l o b i n

A s c e n d i n g E l u t i o n

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

6 0 0 0

0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0

F r a c t i o n N u m b e r

HP

LC

Peak A

rea (

arb

. u

nit

s)

L y s o z y m e

M y o g l o b i n + A p o M y o g l o b i n



Elution control monitoring during run



Descending Elution - Myoglobin



Summary – Batch v. i-TMB

Centrifuge* g/hour g/day

1 litre CPC Batch 0.14 3.24

i-TMB 0.79 19.01

12.5 litre CPC Batch 1.69 40.50

i-TMB 9.90 237.60

* Non-Optimised



Conclusions

• Rapid scale up from the test tube (Hubbuch) to pilot scale (kg/day) is feasible for model systems in batch and continuous process mode

• Now needs evaluating with real systems– Paula Rosa, Lisbon, Portugal (BPP2007)

– Emma Bourton, Brunel, UK (PhD – Syngenta)

– Other volunteers welcome



Challenges

• New liquid-liquid phase systems– Ionic liquids (QUILLS)

– ATPS with higher solubility (Hans-Olef Johansson, Sao Paulo, Brazil)

• Increased sample concentrations in ATPS

– Non-equilibrium sample loading & extraction (NESLE)

– New continuous tube hydrostatic approach

• Development of small footprint, versatile, multi-product facilities for “niche markets” highlighted by Dr Dana Andersen of Genentech

– Initially for high value added products

• UK Government – future emphasis on basic and “user-driven” research

novel liquid-liquid scalable extraction/chromatography technology · 2016-10-05 · optimising...

Documents