AbstractAdvantages of simulated moving bed chromatography (SMBC) over standard lin-

ear or batch processes include dramatically increased productivity, purity, and efficien-cies in chromatography media and buffer utilization. However, complex equipment and multivariate SMBC process development has inhibited universal acceptance and application of this highly efficient purification technology. We have combined a bench-scale multicolumn chromatography system with a comprehensive simulation software toolbox to simplify the design, optimization and demonstration of continuous chroma-tography processes. The software intuitively combines stepwise numerical graphical outputs with flexible fine control options. The dynamic modeling employs the adaptive mesh refinement algorithm for spatial discretization to present robust and accurate simulations of well-known continuous chromatography processes such as SMB, Inter-mittent SMB, and Bio-chromatography in preparative and continuous operation modes. The automated eight column chromatography system performs SMBC and other con-tinuous protocols using software activated pneumatic valves. Fluid flow is controlled by up to eight pumps that can be run individually or in any combination. The step-wise development of SMBC purification for the API precursor 2-hydroxybutyric acid from modeling through the demonstrated continuous purification will be presented.

PurposeTo develop an efficient SMBC separation of the API precursor (R)-2-hydroxybutryic

acid using ChromWorks dynamic modeling software and the Semba Octave Chroma-tography System.

Background

Modeled simulated moving bed purification of 2-hydroxybutyric acidAnthony Grabski1*, Shuvendu Das3, Bruce Thalley1, Jay Yun2, Alla Zilberman1, Soo-iI Kim2, Mani Subramanian3, and Robert Mierendorf1

1Semba Biosciences, Inc., 505 South Rosa Road, Madison, WI 53719 USA2ChromWorks, Inc., 101 Middlesex Tpke, Ste. 6, Burlington, MA 01803 USA

3Center for Biocatalysis and Bioprocessing, University of Iowa, Iowa City, IA 52242 USA

Figure 2. Isocratic SMBC configuration

Figure 3. Glycolate Oxidase (GO) catalyzed oxidation of (RS)-2-hydroxy ac-ids (1). The product, 2-keto acids formed from (S)-enantiomers of 1, is desig-nated as 2.

Figure 4. HPLC profile and rate of conversion of 2-HBA to 2-KBA.

Figure 1. OctaveTM Chromatography SystemVersatile bench top 8-column systemFour to eight pumps for multi-solvent protocolsCapable of performing SMBC and other continuous chromatography protocolsSuitable for gram-to-kilogram scale purificationFlow path options available for biological and chemical applicationsScalable from 12 ml/min to 300 ml/min flow rates

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ReferencesAbel, S. and Juza, M. (2007) in “Chiral Separation Techniques: A Practical Approach,”

G. Subramanian, Ed., Chapter 7, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany.

Das, S., Glenn IV, J. and Subramanian, M. (2010) Biotechnol. Prog. 26, 607-615.

Migliorini, C., Mazzotti, M., Zenoni, G. and Morbidelli, M. (2002) AIChE J. 48, 69-77.

Pedeferri, M., Zenoni, G., Mazzotti, M.and Morbidelli, M. (1999) Chem. Eng. Sci. 54, 3735-3748.

Rajendran, A., Paredes, G. and Mazzotti, M. (2009) J. Chromatogr. A 1216, 709-738.

Seidel-Morgenstern, A. (2004) J. Chromatogr. A 1037, 255-272.

ChromWorks Simulation Software FeaturesEnables modeling of various continuous chromatography processes: 3- and 4-zone SMB, intermittent and sequential SMB, ternary separations and biochromatographyProcess design supported by Triangle TheoryIsotherm parameter estimation and operation simulation

Determination of Modeling ParametersGeometric properties of the adsorbent: rp, particle radius; e, interstitial porosity; ep, particle porosityHydrodynamic properties: Dax, axial dispersion coefficientThermodynamic properties: adsorption isothermsKinetic properties: keff, effective mass transfer coefficient; keff, s, lumped mass transfer coef-ficient; kfilm, external mass transfer resistance

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Figure 8. HPLC profile of GO reaction cycle SMBC system feed.

Figure 9. HPLC profiles of (R)-2-HBA SMBC extract and 2-KBA raffinate.

Figure 5. 2-KBA and (R)-2-HBA pulse data and ChromWorks prediction.

Figure 6. Triangle Theory calculations of the SMBC process.

Figure 7. Simulated multi-column profile and predicted performance data.

Results: Analysis of SMBC Fractions

2-KBA

(R)-2-HBA

FEED

EXTRACT RAFFINATE

Modeling

2-HBA = 31 g/L2-KBA = 1 g/L

2-HBA = 8.5 g/L2-HBA purity >99%2-HBA recovery 75%

2-KBA = 0.15 g/L2-KBA purity 60%2-KBA recovery >98%

Conclusions• Bench-scale SMBC purification was developed to separate the API precursor

(R)-2-HBA from 2-KBA using a crude GO-catalyzed reaction as feed.

• Triangle theory based simulation software facilitated SMBC method development.

• Modeled separation parameters correlated with experimental results to produce (R)-2-HBA with >99% purity.

• These results demonstrate that biocatalytic production and SMBC purification can be used to efficiently produce high-value alpha-hydroxy acid enantiomers.