Sarah Schreiber, Thomas Scheper, Sascha Beutel

Institute of Technical Chemistry, Leibniz University Hannover, Callinstr. 5, D-30167 Hannover

Membrane Ion Exchange Chromatography forPurification of Candida Antarctica lipase B

Center for Applied ChemistryInstitute of Technical ChemistryCallinstr. 5 , 30167 Hannover

Figure 1: Crystal structure of the lipase CalB. Highlighted is the catalytic triad in the active site. Two mobile helices can be found close to the active site [1].

Lipases (EC 3.1.1.3) are enzymes that catalyze the hydrolysis of long chain triacylglycerides into glycerol and free fatty acids. Their structure displays an α/β-hydrolase fold, forming the catalytic triad that binds the substrate (Figure 1). Due to their high stereo- and regioselectivity, but also to the large range of substrates, they open new pathways in the synthesis of structured lipids. The lipase Candida antarctica lipase B (CalB) is one of the most used biocatalysts (e. g. manufacturing of pharmaceuticals, bulk chemicals and food applications). CalB has a pI of 6.0, but it shows an unusual pH profile with a broad isoelectric region from pH 4.0 to 8.0 [2] . This special feature makes the purification of CalB challenging.

Introduction

Disposable membrane adsorbers (MA) are replacing traditional flow-through chomatography more and more, because protein transport is fulfilled by convection while pore diffusion is minimal. Higher flow rates, reduced buffer consumption, shorter process times are some more benefits of this technique. One main topic in modern biotechnology are continuous processes. Several advantages, e. g. significant increase in productivity, greater flexibility, cost reduction and improved product quality, can be named. A continuous set-up with several MAs was build to purify the recombinantly produced CalB (E. coli).

Periodic counter-current (PCC) chromatography

Load Load

Load Load Load

Load Load Load

LoadWash

Wash

Wash

ReceiveWash

ReceiveWash

ReceiveWash

EluteRegenEquil

EluteRegenEquil

EluteRegenEquil

Membrane Adsorber 1

Membrane Adsorber 2

Membrane Adsorber 3

1 2 3 4 5 6

Figure 2: Schematic diagram of the three-column PCC cycle, adapted from [3].

· Continuous bind-and-elute purification process· Colums operate in series configuration· Column switching strategy for feed, wash and elution· Target protein breakthrough from one MA is captured on second MA· Wash is recovered· Set-up provides virtually closed system ® no loss of target protein· Protein loading time ³ recovery and regeneration time· Reduced buffer consumption and higher resin capacity utilization· Suitable to adapt many purification problems

References

[1] Uppenberg et. al. (1994) Structure 15, S. 293–308 [2] Trodler et. al. (2008) Journal of chromatography A 1179,S. 161–167 [3] Godawat et. al. (2012) Biotechnology journal 7 (12), S. 1496–1508

Configuration of the PCC device

Pump

Mixing chamber

Valves

Flow through cuvette

Fraction collector

UV spectrometers

Light source

Membrane adsorber

PCC can be used with conventional bead resins or with membrane adsorbers. Nevertheless, MAs have some main advantages. Mass transfer is realized by convection which leads to higher flow rates up to 40 CV/min. This results in reduced process time. The purification of low protein content is feasible. Furthermore, MAs are easy to scale up by increasing the membrane surface. 21 valves were build up to implement the column switching strategy for 3 MAs. With flow-through cuvettes the protein content at 280 nm can be detected at the outlet of each MA with UV spectrometer. A fraction collector is used to collect the target protein.

Results and Conclusion.

0 1 2 3 4 5 6 M

CalB

180

100

55

40

35

25

10

kDA

Figure 4: Chromatogram of CIEX (left). Equilibration buffer pH 3; Elution buffer pH 6 [2]; High salt wash 1 M NaCl; Flow rate: 5 mL/min. Silver stained SDS-PAGE (right). 0: zero sample; 1-2: Load; 3-5: first peak with pH gradient; 6: second peak with high salt wash; M: unstained protein ladder

ü Recombinant production of CalB in E. coliü First approach: Purification of CalB with cationic

· Optimization of CalB purification· Determination of breakthrough curves· Column switching strategy· Long-term continuous operation

membrane adsorberü Development of a flexible PCC device