Combination of Enantioselective Preparative Chromatography and

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Combination of enantioselective preparative chromatography and racemization: Experimental demonstration and model based process optimization Katarzyna Wrzosek, Isabel Harriehausen, and Andreas Seidel-Morgenstern Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.8b00254 • Publication Date (Web): 13 Nov 2018 Downloaded from http://pubs.acs.org on November 14, 2018

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Organic Process Research & Development

Combination of enantioselective preparative chromatography and racemization: Experimental demonstration and model based process optimization Katarzyna Wrzosek1*, Isabel Harriehausen1, Andreas Seidel-Morgenstern 1,2 1Max‐Planck

Institute for Dynamics of Complex Technical System, Physical and Chemical

Foundations of Process Engineering, Magdeburg, Germany 2 Otto

von Guericke University, Chemical Process Engineering, Magdeburg, Germany

Corresponding author: [email protected], +49 391 6110 321

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Organic Process Research & Development 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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ABSTRACT: Conventional enantioselective preparative chromatographic separation using columns packed with chiral stationary phase is characterized by a 50% yield constraint. Racemization of the undesired enantiomer and recycling the formed mixture is an attractive option to tackle this limit. To implement this concept, potential is seen in particular in applying enzymes immobilized in a second fixed bed. However, the identification of suitable operating conditions and the direct connection of a chromatographic column and an enzymatic reactor is not trivial. The paper presents results of an experimental study applying jointly a batch-wise operated chiral Chirobiotic T column to resolve the two enantiomers of mandelic acid (MA) and a mandelate racemase immobilized on Eupergit CM. The general concept could be successfully demonstrated over several cycles focusing on the provision of (S)-MA. A mathematical model was developed in order to illustrate essential process features and to quantitatively describe the coupled separation and racemization processes. The key ingredients of this model, namely the adsorption isotherms of the two enantiomers on the chiral column and the rate of racemization in the enzymatic reactor, were determined experimentally. The potential of applying the model for further process optimization and generalization is indicated. Keywords: enantioselective chromatography, equilibrium dispersion model, racemization, enzymatic reactor, mandelate racemase 1. INTRODUCTION Enantiomers of a given molecule can have distinctly different effects in living organisms. Therefore, the chirality of a product is of great importance, especially for the pharmaceutical industry. In recent years, there is an increasing demand for highly pure single enantiomers. For certain target products, this demand can be met by stereoselective synthesis. However, in many cases the most straightforward strategy is to synthetize a racemic mixture. In this case, the enantiomers have to be subsequently separated, which is a challenging task due to their virtually 3 ACS Paragon Plus Environment


identical physical properties. The two most important enantioselective separation techniques are crystallization and chromatography, as revised recently.1 While the relatively inexpensive preferential crystallization is often the method of choice for conglomerate forming substances, enantioselective chromatography using chiral stationary phases (CSP) is a highly versatile technique capable of separating the majority of racemates, including those which cannot be easily resolved by crystallization. This versatility is due to a large number of available CSP offering various resolution mechanisms. 2–4 The downside of a production process ending in the separation of the racemate is the related 50% yield limitation. Often the by-product enantiomer is discarded. A number of strategies intended to increase yield and productivity of enantioselective chromatography have been discussed by Kaspereit et al..5 One attractive option is the incorporation of an additional racemization step combined with recycling of the unwanted enantiomer. The mild reaction conditions, facilitating the integration with chromatographic separation, are an important advantage for the use of biocatalysts for the racemization step. The concept of integration of continuous chromatography with racemization reaction was analyzed in detail in theoretical studies, proposing various process designs addressing different purity requirements.6–8 The idea of direct integration of an enzymatic racemization reactor into one process scheme with continuous multi-column chromatography was studied in detail and successfully applied by Bechtold et al.9 One of the advanced concepts, exploiting simulated moving bed separation (SMB) and pH-driven racemization, was implemented for the production of chlorthalidone.7 Later, the separation process of chlorthalidone was also realized in a single column combined with racemization and nanofiltration units.10 Von Langermann et al. reported a process for the separation of the 2′,6′-Pipecoloxylidide involving steady-state-recycling chromatography, crystallization and racemization with Shvo’s catalyst, where the product was obtained with >99.5% enantiopurity.11 The potential of chromatographic separation combined with enzymatic reaction was reported to be also very useful beyond enantiomers separation. For 4 ACS Paragon Plus Environment

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example a SMB setup combined with enzymatic reactor and nanofiltration was successfully applied to increase the yield of producing rare sugars.12,13 A preliminary investigation of the implementation of amino acid racemase in the separation of the methionine enantiomers was presented by Würges et al. and Petruševska-Seebach et al..14,15 The separation of methionine enantiomers was investigated recently and a fully integrated process involving SMB chromatography, enzymatic racemization and nanofiltration was successfully implemented.16 Despite the reports mentioned, examples of implementing the concept of coupled chromatographic separation and enzymatic racemization are still rare. In our recent paper, an enzymatic fixed-bed reactor with immobilized mandelate racemase capable of racemizing the enantiomers of mandelic acid (MA) was developed.17 Mild reaction conditions make enzymatic racemization especially suitable for direct coupling with chromatographic separation and high selectivity of the enzyme ensures that no side reactions appear. A mobile phase, compatible with both racemization reaction and chromatographic separation, was found and the performance of the racemization reactor could be well predicted for steady-state conditions using a rate model.17 Below, we report a successful experimental demonstration of coupling the racemization reactor with single column batch chromatography. Another focus is set on the quantitative description of the chromatographic separation process. Knowledge of the adsorption isotherms of the MA enantiomers will be used to predict the column elution profiles, thus, the inlet profiles for the racemization reactor. Equilibrium dispersion model was used for simulation of the concentration profiles in both the chromatographic column and enzymatic reactor.18,19 Based on the model, suitable fractionation times for product collection and recycling were determined. The effects of the cycle time, the performance of the racemization reactor and the injection volume on the productivity and yield achievable with the overall process were studied. The process model can be used to identify optimal operating conditions. 2. MATERIALS AND METHODS



2.1 SEMI-PREPARATIVE CHROMATOGRAPHY A semi-preparative Chirobiotic T column (15 µm, L=150 mm, d=10mm, Sigma Supelco, St. Louis, USA) was used in the demonstration experiment for separation of (S)- and (R)-MA (Sigma-Aldrich, St. Luis, USA). 20 mM HEPES, 3.3 mM MgCl2 pH 6.8 with 20% methanol was used as a mobile phase for both the semi-preparative Chirobiotic T column and the fixed bed enzymatic reactor connected to the Agilent 1100 HPLC system.17 The signal was recorded with a diode array detector from Agilent at 245 nm and with an advanced laser polarimeter (PDR-Separations., Palm Beach Gardens, USA). The wavelength for the UV signal detection was chosen due to a lower signal compared to the 254 nm used for the analytical measurements (section 2.3) and a higher linear range of the detection. Adsorption isotherms were determined using the fitting method on Agilent 1260 HPLC system (Agilent, Palo Alto, USA) with a diode array detector. 2.2 ENZYMATIC FIXED BED REACTOR Mandelate racemase was expressed in E. coli. Crude extract was immobilized on Eupergit CM with 50-300 μm particle size (Sigma-Aldrich, St.Luis, USA) by 72h coupling in 1M HEPES buffer pH 8.2. Both steps were described in detail by Wrzosek et al..17 The immobilized enzyme was packed in a Tricorn 5/20 column (GE Healthcare Life Sciences, Uppsala, Sweden) to the final bed height Lr = 2.55 cm and maximum operating pressure of 100 bar. Reactor size was chosen based on the activity of the immobilized enzyme, investigated in details in our previous publication17. The flow rate of 0.5 ml/min assured a sufficient residence times for racemization of concentration profiles exiting the chromatographic column. The fixed-bed reactor was connected to the Agilent 1100 HPLC system (Agilent, Palo Alto, USA) during the proof-of-concept experiment runs (see 4.2.). 2.3 ANALYTICAL CHROMATOGRAPHY The concentration of MA enantiomers was determined with an analytical Chirobiotic T column (5 µm, L=150 mm, d=4.6mm, Sigma Supelco, St. Louis, USA) with 0.3 M TEAAv (triethylammonium acetate) buffer pH 4.02 with 20% methanol as a mobile phase. Flow rate of 0.5 mL/min and an 6 ACS Paragon Plus Environment

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injection volume of 5 µl, or 15 µl for very diluted samples, were used. Measurements were performed on Agilent 1260 HPLC system with UV detection at 254 nm. The wavelength with a higher signal for a given concentration of MA was chosen to facilitate the detection of profiles for very diluted samples. 2.4 EXPERIMENT SETUP FOR THE PROCESS COMBINATION A proof-of-concept experiment was designed for the production of (S)-MA as a selected target enantiomer to show the applicability of chromatographic separation coupled with by-product racemization. The setup used is shown in figure 1. Initially, an injected amount of a racemic mixture is separated on the semi-preparative Chirobiotic T chromatographic column. The first peak exiting the column, i.e. the product (S)-MA, is directed to the detectors via a switch valve V1. Next, it is periodically directed through a switch valve V2 to the product collection tank as fraction F1. In this first phase of the process there is no flow through the enzymatic reactor. Next as the second peak, containing the by-product (R)-MA, elutes at the outlet of the chromatographic column, it is directed via valve V1 to the fixed bed enzymatic reactor. The racemized by-product stream leaving the reactor is directed to the detectors and periodically collected after the switch valve V2. This racemized fraction F2 is then recycled and mixed into the feed tank from which samples for the next cycle are injected periodically.

Figure 1. Flow scheme for the experiment employing chromatographic separation and byproduct recycling after enzymatic racemization. The (S)-MA (selected target) is marked in red, 7 ACS Paragon Plus Environment


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(R)-MA in blue, the racemic mixture in purple. These colours will be used later again in figures 4, 6 and 7 showing elution profiles. In the experiments 100 µL sample volumes of the racemic mixture were injected periodically from a feed tank with an initial liquid phase volume of 50 mL and concentration of 40 g/L.

3. THEORY For process simulation, the separation column and the racemization reactor were described by the equilibrium dispersion model. The adsorption behaviour of R- and (S)-MA was described with first and second order adsorption isotherm model and the enzymatic racemization with a reversible Michaelis-Menten kinetic equation. Additionally, the dynamics of the feed tank were considered. Finally, suitable process evaluation parameters are introduced. 3.1 EQUILIBRIUM DISPERSIVE MODEL The migration of the concentration fronts in the chromatographic column is based on the mass balance shown in equation (1). This equilibrium dispersion model considers the liquid and solid phase as well as convective mass transfer together with a dispersion term.19 𝜕𝑐𝑖𝑐 𝜕𝑡

𝜕2 𝑐𝑖𝑐

𝑐 = 𝐷𝑎𝑝𝑝 ∙

𝜕𝑧 2

− 𝐹𝑐 ∙

𝜕𝑞𝑖 (𝑐1𝑐 ,𝑐2𝑐 ) 𝜕𝑡

− 𝑢𝑐 ∙

𝜕𝑐𝑖𝑐 𝜕𝑧

𝑖 = 1,2

(1)

Here, 𝑐𝑖 is the concentration of the component 𝑖 in the mobile phase, 𝑞𝑖 is the loading (adsorbed component on the solid phase), 𝑡 is the time variable, 𝑧 the space variable, Fc is the phase ratio in the chromatographic column and uc is the interstitial linear velocity. The superscript 𝑐 indicates 𝑐 is the apparent dispersion coefficient parameters of the chromatographic column. 𝐷𝑎𝑝𝑝

𝑐 𝐷𝑎𝑝𝑝 =

𝑢𝑐 ∙𝐿𝑐 2 ∙ 𝑁𝑃𝑐

=

𝑢𝑐 ∙ 𝐻𝐸𝑃𝑇 𝑐 2

(2)

with

𝐻𝐸𝑇𝑃𝑐 =

𝐿𝑐 𝑁𝑝𝑐

(3)


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The dispersion coefficient is related to the number of theoretical plates, 𝑁𝑝𝑐 and the column length, 𝐿𝑐 . Dispersion can also be quantified using the height of an equivalent theoretical plate in the chromatographic column, 𝐻𝐸𝑇𝑃𝑐 . 𝐹 𝑐 is defined as

𝐹𝑐 =

1 − 𝜖𝑡𝑐 𝜖𝑡𝑐

(4)

where 𝜖𝑡𝑐 is the total column porosity. The column is assumed to be unloaded prior to the first injection: 𝑐𝑖𝑐 (𝑡 = 0, 𝑧) = 0

for

0 ≤ 𝑧 ≤ 𝐿𝑐 ,

𝑘= 1

(5)

Racemic mixture with the concentration 𝑐𝑖𝑡𝑎𝑛𝑘 is periodically injected in 𝑁𝑐𝑦𝑐 rectangular pulses from the feed tank onto the column. This cycle time, ∆𝑡𝑐𝑦𝑐 , is the time difference between two 𝑘 𝑘+1 consecutive injections 𝑡𝑖𝑛𝑗 and 𝑡𝑖𝑛𝑗 :

𝑘+1 𝑘 ∆𝑡𝑐𝑦𝑐 = 𝑡𝑖𝑛𝑗 − 𝑡𝑖𝑛𝑗

with

𝑘 = 1 … 𝑁𝑐𝑦𝑐

(6)

Cycle times shorter than the retention times of the components might possess potential to eventually improve the productivity. This results in the following boundary conditions for the column inlet:

𝑐𝑖𝑐,𝑖𝑛 = {

𝑐𝑖𝑡𝑎𝑛𝑘 , 0

0 + ∆𝑡𝑐𝑦𝑐 ∙ (𝑘 − 1 ) < 𝑡 < ∆𝑡𝑖𝑛𝑗 + ∆𝑡𝑐𝑦𝑐 ∙ (𝑘 − 1) k=1 …𝑁𝑐𝑦𝑐 𝑒𝑙𝑠𝑒 ,

(7)

For the reactor outlet the Danckwerts boundary conditions 21 were applied: 𝜕𝑐𝑖𝑐

|

𝜕𝑧 𝑡,𝑧=𝐿𝑐

(8)

=0

where 𝑐𝑖𝑐,𝑖𝑛 is the inlet concentration into the chromatographic column and ∆𝑡𝑖𝑛𝑗 is the length of each injection. Equation (1) was solved numerically using a standard explicit finite-difference method 18,20: 9 ACS Paragon Plus Environment


𝑐 𝑚 𝑐𝑖𝑐 |𝑚 𝑛+1 = 𝑐𝑖 |𝑛 −

Δ𝑧 uc ∙ Δ𝑡

𝑐 𝑚−1 ] 𝑚−1 [ 𝐹 𝑐 (𝑞𝑖 |𝑚 )+ 𝑐𝑖𝑐 |𝑚 𝑛 − 𝑞𝑖 |𝑛 𝑛 − 𝑐𝑖 |𝑛

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(9)

In this equation, m and n are the running variables in time and space, respectively. The dispersion effect is incorporated by matching the physical and numerical dispersions respecting eq. (2). This specifies appropriate grid sizes in space ∆𝑧 and time ∆𝑡: (10)

∆𝑧 = 𝐻𝐸𝑇𝑃𝑐 2 ∙ 𝐿𝑐

∆𝑡 = 𝑁𝑐 ∙𝑢𝑐 = 𝑃

2 ∙ 𝐻𝐸𝑇𝑃𝑐 𝑢𝑐

(11)

Stability of the simulation is guaranteed fulfilling the Courant-Friedrichs-Lewy condition:

𝑎𝑐 = 𝑢̅𝑖,𝑚𝑖𝑛 ∙

∆𝑡 ∆𝑧

≥ 1 with

𝑢̅𝑖,𝑚𝑖𝑛 =

𝑢𝑐 ̅𝑖 1 +𝐹 𝑐 ∙𝐻

(12)

with 𝑎𝑐 being the Courant number 𝑢̅𝑖,𝑚𝑖𝑛 being the mean velocity of the component 𝑖 under linear ̅. conditions characterized by an averaged Henry constant 𝐻 3.2 ADSORPTION BEHAVIOUR The simplest model to describe the adsorption behaviour of a component 𝑖 is the linear adsorption isotherms model: (13)

𝑞𝑖 = 𝐻𝑖 ∙ 𝑐𝑖 where 𝐻𝑖 is the Henry coefficient.

A more complex model capable of describing often encountered complex equilibrium behaviour, (e.g. Langmuir or anti-Langmuir) and isotherms with inflection points, is the quadratic adsorption isotherm:

𝑞𝑖 = 𝑞𝑠𝑎𝑡,𝑖

𝑐𝑖 (𝑏𝑖,1 +2∙𝑏𝑖,2 ∙ 𝑐𝑖 )

(14)

1+𝑏𝑖,1 ∙𝑐𝑖 +𝑏𝑖,2 ∙𝑐𝑖2

Where 𝑞𝑠𝑎𝑡,𝑖 is the saturation capacity of the column, 𝑏𝑖,1 and 𝑏𝑖,2 are adsorption parameters. These adsorption isotherm parameters were determined with the peak fitting method18, based on 10 ACS Paragon Plus Environment

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𝑒𝑥𝑝

experimentally obtained elution profiles 𝑐𝑖

of single component pulse injections with varying

concentrations. With this method, suitable free parameters can be optimized by minimizing an objective function OF (eq. (15)) using a least square solver.

𝑂𝐹 =

2

𝑁

∑𝑗 𝑒𝑥𝑝 (𝑐𝑖𝑚𝑜𝑑𝑒𝑙 (𝑡, 𝑧 = 𝐿𝑐 ) − 𝑐𝑖𝑒𝑥𝑝 (𝑡, 𝑧 = 𝐿𝑐 )) → Min

for j = 1 … Nexp

(15)

Here, 𝑐𝑖𝑚𝑜𝑑𝑒𝑙 are the eluting profiles predicted by the model and 𝑁𝑒𝑥𝑝 is the number of different consecutively injected and jointly analysed elution profiles. 3.3 FRACTIONATION TIME DETERMINATION Criteria for specifying the fractionation times 𝑡1𝑐 , 𝑡2𝑐 and 𝑡3𝑐 have to be defined to direct the first fraction F1 (𝑡1𝑐 - 𝑡2𝑐 ) with the target enantiomer E1 to the product tank and to send the second fraction F2 (𝑡2𝑐 - 𝑡3𝑐 ) for recycling to the racemization column. Hereby, 𝑡1𝑐 is the time when the first component is eluting with a concentration above the threshold concentration 𝑐𝑇ℎ𝑟𝑒𝑠 . To meet the purity requirements in each cycle, the target enantiomer (E1) can only be collected as long as the overall contamination of E2 in F1 remains below an acceptable impurity level, 1%. The time 𝑡2𝑐 characterizes the point where this requirement is no longer fulfilled or where E1 starts to elute with a concentration below 𝑐𝑇ℎ𝑟𝑒𝑠 again. The second fraction starts at the just specified time 𝑡2𝑐 and ends at 𝑡3𝑐 when the concentration of E2 drops below the threshold concentration. It mainly contains the by-product enantiomer and is therefore forwarded to the racemization reactor. For frequent injections, 𝑡3𝑐 (𝑘) also marked the start of the next cycle 𝑡1𝑐 (𝑘 + 1). Within the time span 𝑡2𝑟 - 𝑡3𝑟 the racemized fraction (F2’) elutes from the fixed bed reactor and is recycled to the feed tank. In summary: 

F1: 𝑡1𝑐 – 𝑡2𝑐

fraction contains target E1 with 𝑃𝑈𝑅1 ≤ 99%  product tank



F2: 𝑡2𝑐 – 𝑡3𝑐

fraction contains E2 (and eventually partly E1 ) recycling column



F2’: 𝑡2𝑟 – 𝑡3𝑟

racemized fraction F2


 feed tank


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3.4 MODEL FOR RACEMIZATION REACTOR The isothermal mass balance for the racemisation reactor (marked by superscript r), eq. (16), originates from equation (1), after eliminating the solid phase storage term and adding a reaction term. The latter included the reactor phase ratio 𝐹 𝑟 , the stoichiometric coefficients 𝜈𝑖 and the reaction rate 𝑟𝑟𝑎𝑐 : 𝜕𝑐𝑖𝑟 𝜕𝑡

𝑟 = 𝐷𝑎𝑝𝑝 ∙

𝜕2 𝑐𝑖𝑟 𝜕𝑧 2

− 𝑢𝑟 ∙

𝜕𝑐𝑖𝑟 𝜕𝑧

− 𝐹 𝑟 ∙ 𝜈𝑖 ∙ 𝑟𝑟𝑎𝑐

i = 1,2

(16)

𝑟 Analogous to the chromatographic column, 𝐷𝑎𝑝𝑝 is a corresponding apparent dispersion.

Figure 1 shows, that only F2 is directed to the racemization reactor, while F1 is sent to the product tank. Thus, there is no convective mass transfer in the reactor during the time 𝑡1𝑐 – 𝑡2𝑐 when volumetric flow rate in the reactor 𝑉 𝑟̇ equals zero: 0 , 𝑉̇ 𝑟 = { ̇ 𝑐 𝑉 ,

𝑡1𝑐 (𝑘) < 𝑡 < 𝑡1𝑐 (𝑘) 𝑡2𝑐 (𝑘) < 𝑡 < 𝑡3𝑐 (𝑘)

(17)

In this time span the apparent dispersion is reduced to the molar dispersion 22: 𝑉̇ 𝑟̇ =0

𝑟 𝑟 = 𝐷𝑚𝑜𝑙 + 𝛾 𝑢𝑟 𝑑𝑝 ⇒ 𝐷𝑎𝑝𝑝

𝑟 𝑟 = 𝐷𝑚𝑜𝑙 𝐷𝑎𝑝𝑝

(18)

with 𝛾 being a weighting factor and 𝑑𝑝 the particle diameter. In this work, the molar dispersion of 𝑟 the liquid phase in the parking state was assumed to be 𝐷𝑚𝑜𝑙 = 10−9

𝑚2 23 . 𝑠

The racemization reactor is also considered to be filled initially only with mobile phase. 𝑐𝑖𝑟 (𝑡 = 0, 𝑧) = 0

for 0 ≤ 𝑧 ≤ 𝐿𝑟 , 𝑘 = 1

(19)

The elution profile collected as fraction F2 at the outlet of the chromatographic column is directed to the inlet of the reactor in the time frame 𝑡2𝑐 – 𝑡3𝑐 . Thus, the boundary conditions of the reactor are defined as followed:


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𝑐 𝑐 (𝑧 = 𝐿𝑐 ) , 𝑡2𝑐 (𝑘) < 𝑡 < 𝑡3𝑐 (𝑘) 𝑐𝑖𝑟,𝑖𝑛 = { 𝑖 0, 𝑒𝑙𝑠𝑒 𝜕𝑐𝑖𝑟 | 𝜕𝑧 𝑡,𝑧=𝐿𝑟

k=1 …𝑁𝑐𝑦𝑐

(20)

(21)

=0

Here 𝑐𝑖𝑟,𝑖𝑛 is the inlet concentration of the racemisation reactor. Equation (16) is solved numerically with and without convective mass transfer. In the first time period (𝑡1𝑐 – 𝑡2𝑐 ) the discretisation is done analogous to the chromatographic column model (see eq. (9)). While the peak is parked in the column (in the time span 𝑡2𝑐 – 𝑡3𝑐 ), the following discretization, based on a central finite difference approximation, is applied:

= 𝑐𝑖𝑟 |𝑚 𝑐𝑖𝑟 |𝑚+1 𝑛 − 𝑛

𝑟 𝐷𝑚𝑜𝑙 Δ𝑡 Δ𝑧 2

𝑐 𝑚 𝑐 𝑚 [ 𝑐𝑖𝑐 |𝑚 𝑛+1 − 2 𝑐𝑖 |𝑛 + 𝑐𝑖 |𝑛−1 ]

(22)

3.5 ENZYME ACTIVITY AND REACTION RATES The kinetic parameters of the racemization reaction were obtained for the immobilized enzyme as described in detail in

17.

The enzymatic activity can be quantified with reversible Michaelis-

Menten kinetics with assumption of reaction rates being identical in both directions 24:

𝑟𝑟𝑎𝑐 = 𝑟𝑚𝑎𝑥

(𝑐1 −𝑐2 ) 𝐾𝑀 +𝑐1 +𝑐2

(23)

In eq. (23), 𝑟𝑚𝑎𝑥 is the maximal reaction rate and 𝐾𝑀 is the Michaelis-Menten coefficient. 3.6 BALANCE OF THE FEED TANK Due to periodical removal of feed from the tank and addition of recycled stream, masses and concentrations in the feed tank fluctuate. The following mass balance captures this: 𝑑𝑚𝑖𝑡𝑎𝑛𝑘 𝑑𝑡

= 𝑉𝑡𝑎𝑛𝑘 (𝑡) ∙

𝑑𝑐𝑖𝑡𝑎𝑛𝑘 𝑑𝑡

= 𝑉 𝑐̇ ∙ (𝑐𝑖𝑡𝑎𝑛𝑘,𝑖𝑛 (𝑡) − 𝑐𝑖𝑐,𝑖𝑛 (𝑡))

with


(24)


𝑐𝑖𝑡𝑎𝑛𝑘,𝑖𝑛 (𝑡) = 𝑐𝑖𝑟 (𝑡, 𝑧 = 𝐿𝑟 )

Page 14 of 29

(25)

with the volumetric flow rate 𝑉 𝑐̇ and the superscript tank indicating variables and parameter of the feed tank. Integrating over one cycle, the mass removed, 𝛥𝑚𝑖𝑡𝑎𝑛𝑘 , can be estimated with equation (26): 𝑡 𝑘+1

𝛥𝑚𝑖𝑡𝑎𝑛𝑘 = 𝑉 𝑐̇ ∙ ∫ 𝑘𝑖𝑛𝑗 (𝑐𝑖𝑡𝑎𝑛𝑘,𝑖𝑛 (𝑡) − 𝑐𝑖𝑐,𝑖𝑛 (𝑡)) 𝑑𝑡 𝑡

(26)

𝑖𝑛𝑗

3.7 PROCESS EVALUATION PARAMETER Purity parameters need to be fulfilled for the collection of E1 in the fraction F1. The purity of fraction F1 can be calculated with the collected mass of both components 𝑖 between 𝑡1𝑐 and 𝑡2𝑐 : 𝑡𝑐

𝑚𝑖𝑐 = 𝑉 𝑐̇ ∙ ∫𝑡 𝑐2 𝑐𝑖𝑐 (𝑡, 𝑧 = 𝐿𝑐 ) 𝑑𝑡

(27)

1

𝑃𝑈𝑅1 =

𝑚𝑖𝑐 𝑚1𝑐 + 𝑚2𝑐

(28)

∙ 100%

Typically, a specific product purity 𝑃𝑈𝑅1 is required, e.g. 99%, for many industrial applications. Furthermore, to be economically efficient, a process should be optimized with respect to its productivity, considering here the separation is the rate-determining step of the coupled process. The productivity of providing the first eluting enantiomer, 𝑃𝑟1 , can be calculated as follows:

𝑃𝑟1 =

𝑚1𝑐 ∆𝑡𝑐𝑦𝑐 ∙ 𝑉 𝑐

=

𝑉 𝑐̇ ∆𝑡𝑐𝑦𝑐 ∙ 𝑉 𝑐

𝑡𝑐

∙ ∫𝑡 𝑐2 𝑐1𝑐 (𝑡, 𝑧 = 𝐿𝑐 ) 𝑑𝑡 1

𝑖 = 1,2

(29)

where 𝑚1𝑐 is the mass of the target component 1 collected in the product tank. In eq. (29) the productivity is scaled to the cycle time, ∆𝑡𝑐𝑦𝑐 , and the volume of the chromatographic column, 𝑉 𝑐 . A third key parameter is the process yield. In this parameter the most significant difference between the coupled process and exclusively carried out chromatographic separation can be seen. For a single injection, 𝑌1𝑐 is the ratio between the collected mass of the target compound, 𝑚1𝑐 , and the mass injected onto the chromatographic column, 𝑚1𝑐,𝑖𝑛 , 𝑚2𝑐,𝑖𝑛 . 14 ACS Paragon Plus Environment

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𝑡 𝑘+1

𝑌1𝑐 = ∫ 𝑘𝑖𝑛𝑗 𝑡 𝑖𝑛𝑗

𝑐1𝑐 (𝑡,𝑧=𝐿𝑐 ) 𝑖𝑛 𝑐1 (𝑡)+𝑐2𝑖𝑛 (𝑡)

=

𝑚1𝑐 𝑚1𝑐,𝑖𝑛

+ 𝑚2𝑐,𝑖𝑛

(30)

In the coupled process, the racemized second fraction is recycled and thus a yield 𝑌1𝑡𝑜𝑡 of 100% can be obtained. 4. RESULTS AND DISCUSSION 4.1 CHROMATOGRAPHIC SEPARATION: PRELIMINARY EXPERIMENTAL RESULTS The mobile phase used in all experiments consisted of 20 mM HEPES 3.3 mM MgCl2 pH 6.8 with 20% methanol. It was selected in accordance with our previous study to facilitate both chromatographic separation and enzymatic racemization steps 17. In order to evaluate the effect of the amount of racemic (RS)-MA feed, 100 µL sample volumes were injected with concentrations of 20 g/L, 40 g/L and 80 g/L. The corresponding elution profiles recorded are shown in figure 2. The largest injection concentration was rejected as most of the eluting peaks exceeded the detection limit of the diode array detector. The injection concentration of 40 g/L (RS)-MA resulted in a near baseline separation of the two enantiomers and was chosen for further investigations. The first eluting (S)-MA was chosen as the target enantiomer. This position in the elution order is usually favorable in preparative chromatography as the first eluting component can be collected with a higher productivity compared to later eluting compounds 25. In order to collect pure (S)MA and racemize and recycle the (R)-MA fraction, the characteristic times introduced in section 3.3 have to be determined. The first two fractionation times, 𝑡1𝑐 and 𝑡2𝑐 , depended only on the separation progress in the chromatographic column. Therefore, they can be identified by analyzing just a chromatogram without the racemization (e.g. the dashed lines in fig. 2 belonging to the black profile), as exploited by Nimmig et al. 10. The end of collecting the recycled second peak, i.e. the fractionation time 𝑡3𝑟 , cannot be identified in this chromatogram since it will be affected by dispersion in the racemization reactor, which is additionally passed. The last 15 ACS Paragon Plus Environment


fractionation time can be obtained during the coupled process experiment as described in section 4.4.

Figure 2. Separation of mandelic acid enantiomers on a semi-preparative Chirobiotic T column. Elution profiles for injection of 100 µL sample of (RS)-MA at 0.5 mL/min flow rate. Total concentrations of injected MA: 20 g/L (light grey), 40 g/L (black) and 80 g/L (dark grey). (S)-MA is eluting first. The two dashed lines indicate fractionation times selected based on the chromatogram for injection of MA with concentration of 40 g/L. 4.2 DEMONSTRATING THE COMBINED PROCESS: SEVERAL CONSECUTIVE CYCLES A proof-of-concept experiment of the combined process was performed following the scheme shown in figure 1. The small feed tank contained initially 50 mL of racemic (RS)-MA with a total concentration of 40 g/L. In each cycle 100 L were injected into the column. In accordance with the determined fractionation times for the second enantiomer, 2.2 mL of racemized by-product fraction was produced and recycled into the feed tank before the next injection was carried out. Since the concentration of the recycled fractions was only 0.91 g/L, this procedure caused a slow gradual dilution in the feed tank (fig. 3). In order to achieve cyclic steady state conditions to be evaluated a recharging of the tank was performed when the concentration dropped below 35 g/L. This was done after the fourth cycle by addition of stock (RS)-MA solution with concentration of 80 g/L which is close to the solubility limit. Obviously, this would not be needed with a larger feed tank. The feed tank dynamics are quantified by eqs. (24)-(26).


Page 16 of 29

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Figure 3. Changes of the (RS)-MA concentration (black) and volume (blue) of the feed tank due to withdrawal for the nine injections performed and periodic return of a diluted racemized byproduct. Points are indicating the concentration of each injection. Dotted lines indicate the maximum and minimum concentrations in the tank and the initial volume in the tank. The chromatogram obtained after the first cycle is shown in figure 4. The signals of the two detectors were used to calculate the concentration profiles. The UV detector signal is proportional to the total MA concentration (regardless of the enantiomer), whereas the polarimeter signal quantifies the enantiomeric excess of the mixture. Based on these two signals, the successful racemization of the second by-product peak can be recognized. Since the racemization reactor contains just solvent at the beginning of every cycle there is a delay between the recorded end of the product peak (𝑡2𝑐 ) and the recorded beginning of the corresponding racemized by-product peak (𝑡2𝑟 ). The purity of the collected product F1 fractions as well as the final composition in the feed tank and the composition of the last racemized by-product F2 fraction were quantified by chiral analytical chromatography. According to the results of this analysis the racemization in the enzymatic reactor was complete. The F1 fractions contained pure (S)-MA with an average concentration of 0.95 g/L. After nine cycles of the periodically operated coupled separation and racemization process, 18 mg of (S)-MA with 99% purity were collected according to the analytical



chromatography profiles of F1 samples. All of the second by-product enantiomer was totally racemized and recycled to the feed tank. The experiment was carried out with no sign of decrease in enzymatic reactor activity or loss of selectivity of the chromatographic column. The experimental work done with the coupled columns took over two weeks with no changes in performance. Several similar studies involving a coupling of chromatographic separation with enzyme membrane reactor reported enzyme deactivation during the runs, which was compensated by using excessive amounts of enzyme in the reactor to safeguard stable operation over time 13,16. The superior stability of the enzyme used in this study is most likely at least partially due to the immobilization of the enzyme in a fixed bed reactor. This decreases the risk of enzyme deactivation by proteolysis, aggregation or shear stress caused by vigorous stirring. 26,27

Figure 4. Chromatograms obtained in first cycle of the proof-of-concept experiment (100 L injection of 40g/L (RS)-MA at 0.5 mL/min flow rate). Concentration change based on polarimeter response is indicated in grey. The profile based on response from diode array detector is shown in black, red ((S)-MA peak) and purple (racemized by-product). Dashed lines indicate the fractionation times.


Page 18 of 29

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4.3 ESTIMATION OF MODEL PARAMETERS An accurate process prediction requires a detailed model, incorporating adsorption isotherms as well as a model for the chromatographic column and the racemization reactor with well estimated model parameters. First, the adsorption equilibrium of the chromatographic column needed to be described. Based on experimental peaks, (R)-MA shows a linear adsorption behaviour within concentration range of the injected and eluted peaks (fig. 5). Thus, a linear isotherm was assumed for this enantiomer (eq. (13)). However, the S-enantiomer peaks display significant fronting, characteristic for antiLangmuir behaviour. Therefore, the adsorption equilibrium of this enantiomer was modelled with the quadratic adsorption isotherm model (eq. (14)). The parameters of both enantiomers were determined using a peak fitting method based on the least square algorithm (eq. (15)). Remaining inconsistencies between predicted and experimental profiles for (R)-MA are a consequence of the limitations of the assumed dispersion equilibrium model, the isotherm model structure and the neglect of competitive adsorption. The consequences of the latter were found to be minor in more extensive simulations including an empirical competitive isotherm model. The differences of the peak maxima between experimental and modelled profiles visible for the peaks belonging to the highest injection concentration, shown in fig. 5 (40 g/L), are also due to approaching of the detection limit of the UV-Vis detector.

Figure 5. Comparison of experimental (solid line) and simulated (dashed line) elution profiles of (S)-MA (black) and (R)-MA (grey) obtained after 100 µL injections of racemate with concentrations of 5, 10, 20, 40 g/L, at flow rate of 0.5 mL/min. The simulated profiles were 19 ACS Paragon Plus Environment


Page 20 of 29

modelled with the equilibrium dispersion model (eq. (1)) and the adsorption parameters in table 1. The dispersion coefficients and 𝐻𝐸𝑇𝑃 of the chromatographic column and the racemization reactor were determined with eq. (2)–(3). The 𝐻𝐸𝑇𝑃𝑟 of the self- packed racemization reactor is over 20 times higher than 𝐻𝐸𝑇𝑃𝑐 , which would result in an equally larger grid size (eq. (3)). Yet, the grid size does not significantly influence the qualitative elution profile of the reactor, but only 𝑟 its resolution. Thus, for the sake of simplicity, the dispersion coefficient 𝐷𝑎𝑝𝑝 was assumed to be 𝑐 equal to 𝐷𝑎𝑝𝑝 (eq. (2)). The Michaelis-Menten parameters were determined and reported in our

previous paper 17. All parameter are listed in table 1. Table. 1. Modelling parameters Parameter Saturation capacity (eq. (14)) Equilibrium isotherm parameter (eq. (14)) Equilibrium isotherm parameter (eq. (14)) Henry coefficient (eq. (13)) Dispersion coefficient in chromatographic column (eq. (2)) Height of an equivalent theoretical plate in the chromatographic column (eq. (3)) Maximal reaction rate (eq. (23)) Michaelis-Menten coefficient (eq. (23)) Dispersion coefficient in racemization reactor (eq. (2)) Height of an equivalent theoretical plate in the racemisation reactor (eq. (3))

qsat,1 b11 b12 H2 𝑐 𝐷𝑎𝑝𝑝

4.70 0.0604 0.0137 0.694 4.81∙ 10−9

g/L L/g L2/g2 m2/s

𝐻𝐸𝑇𝑃𝑐

5.56 ∙ 10−5

m

rmax KM 𝑟 𝐷𝑎𝑝𝑝 𝐻𝐸𝑇𝑃𝑟

169.5 8.59 1.56∙ 10−4 7.49 ∙ 10−3

U*/gcarrier mM m2/s m

* one unit of enzymatic activity U is equal to the amount of enzyme converting 1 µmol of the substrate in 1 minute. 4.4 PROCESS SIMULATION The design of the coupled process depicted in fig. 1 requires a good understanding of development of the peak profiles in the chromatographic column. In order to design the process with maximized productivity, it is important to be able to predict the elution profiles for various process conditions. Equilibrium dispersion model was employed to describe enantiomers migration in the chromatographic column (eq. (1)). Together with adsorption isotherms (eq. (13-14)) the model 20 ACS Paragon Plus Environment

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was able to predict elution profiles with a good accuracy (fig. 5). Next, three basic fractionation times needed to be established: 𝑡1𝑐 for start of collection of fraction 1 (F1), 𝑡2𝑐 for switching valve V2 to direct fraction 2 (F2) into the racemization column and 𝑡3𝑐 to mark the end of F2. The fixed-bed reactor was modelled with eq. (16) and the enzyme reaction kinetics (eq. (23)). For the time span 𝑡1𝑐 – 𝑡2𝑐 the peak parked in the reactor was modelled with eq. (22). Times 𝑡1𝑐 and 𝑡3𝑐 are set based on the concentration threshold (𝑐𝑇ℎ𝑟𝑒𝑠 = 0.02 ∙ 𝑐1𝑡𝑎𝑛𝑘 = 0.04 𝑔/𝐿) above which the collection of the fractions is reasonable (see section 2.5). The fractionation time 𝑡2𝑐 is based on the minimum purity requirement of the product fraction F1. With the simulated elution profiles, the three fractionation times can be easily calculated for a desired racemate load facilitating the experimental design. For initial simulations, the experimental settings described in section 4.2 were used. The only 𝑡𝑏 parameter changed for the simulation was the cycle time, which was reduced to ∆𝑡𝑐𝑦𝑐 = 5.4 min 𝑡𝑏 (tb for touching bands). For ∆𝑡𝑐𝑦𝑐 ≤ 5.4 min, the end of one cycle 𝑡3𝑐 (𝑘) is equivalent to the

collection start of the following cycle 𝑡1𝑐 (𝑘 + 1). The resulting elution profiles are depicted in figure 6. Equivalent to the experiment in section 4.2, fraction F2 with (R)-MA is directed to the racemization reactor (fig. 6c) and (S)-MA is collected in the product tank (fig. 6d). Already the profile corresponding to the second injection is in a quasi-steady state.



Page 22 of 29

Figure 6. Concentration profiles and collected product resulting from injection of a racemic mixture of (RS)-MA (purple) from a feed tank of infinite volume: a) Injections of 100 µL pulses 𝑐,𝑖𝑛 with a concentration 𝑐12 of 40 g/L (RS)-MA resulting in a touching bands resolution with a cycle 𝑡𝑏 time of ∆𝑡𝑐𝑦𝑐 = 5.4 min. b) Elution profiles of S- (red) and R- (blue) mandelic acid at the end of the chromatographic column (CC). c) The elution profiles at the outlet of the racemization reactor (RR). d) The increase of collected amount of (S)-MA with a purity constraint of PUR1≥99%. Figure 7a-d shows the simulation for identical injections with the cycle time reduced to 3.55 min. As can be seen in fig. 8a, this is the cycle time allowing the highest productivity of (S)-MA purification. A reduction of cycle time below 5.4 min causes the bands of two consecutive injections to partially overlap. Thus, the productivity is affected by the decreasing amount of pure collected product. Nevertheless, for (S)-MA, productivity is improved up to a cycle time of 3.55 𝑜𝑝𝑡,𝑆

min, before the loss in mass exceeds the gain in time. For the optimized cycle time, ∆𝑡𝑐𝑦𝑐 , the predicted productivity reaches 56.03 g/L/day, which is 23% more than for the case of touching bands separation depicted in figure 6. By analogy, the elution profiles exiting the chromatographic column (fig. 7b) and the racemization reactor (fig. 7c) are shown as well as collection of the target enantiomer (fig. 7d).


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𝑐,𝑖𝑛 Figure 7. Concentration profiles resulting in injection of a racemic mixture of (RS)-MA (𝑐12 = 40 g/L, 𝑉𝑖𝑛𝑗 = 100 µL). Figures a-d depict a process with (S)-MA as target enantiomer for the 𝑜𝑝𝑡,𝑆 optimized cycle time ∆𝑡𝑐𝑦𝑐 = 3.55 min. Graphs e-h show a process with (R)-MA collected as a 𝑜𝑝𝑡,𝑅 target for ∆𝑡𝑐𝑦𝑐 = 4.62 min. The optimized cycle times provide the highest productivity combined with minimum purity (𝑃𝑈𝑅𝑖 ≥ 99%) of the collected target enantiomer. By analogy to figure 6 the injection (a and e), elution profiles exiting chromatographic column (b and f), elution profiles exiting the racemization reactor (c and g) and amount of collected target and by-product enantiomer (d and h) are shown.

For all cycle times shorter than the cycle times corresponding to the touching bands situation and therefore an overlap of consecutive cycles, the threshold concentration 𝑐𝑇ℎ𝑟𝑒𝑠 was increased to enhance productivity. The same setup can be used to perform the purification of the counter enantiomer, (R)-MA. Because of the anti-Langmuir shape of the S-enantiomer its front strongly contaminates the Renantiomer peak. Thus, the optimal productivity for harvesting (R)-MA requires a longer cycle



time

of

𝑜𝑝𝑡,𝑅

∆𝑡𝑐𝑦𝑐

=

4.62

min

Page 24 of 29

(fig.

7e-h).

Figure 8. Effect of different process variables on productivity and yield: (a) Productivity of separation process, with (S)-MA (black circle) or (R)-MA (grey circle) as the target enantiomer, in 𝒕𝒃 dependence of the cycle time for 𝒄𝒄,𝒊𝒏 𝟏𝟐 = 40 g/L (RS)-MA and 𝑽𝒊𝒏𝒋 =100 µL. At ∆𝒕𝒄𝒚𝒄 = 5.4 min consecutive bands touch. (b). Effect of incomplete racemization, expressed by the effluent 𝒆𝒆𝒓𝟐 on 𝒄,𝒊𝒏 the productivity of (S)-MA for 𝒄𝒄,𝒊𝒏 for the cycle time 𝟏 = 40 g/L and changing concentrations 𝒄𝟐 𝒐𝒑𝒕,𝑺 𝒕𝒃 ∆𝒕𝒄𝒚𝒄 (black rhombus) and ∆𝒕𝒄𝒚𝒄 (hollow rhombus) . (c) Productivity of (S)-MA as a function of 𝒕𝒃 mass of the injected (S)-MA (black triangle) for 𝒄𝒄,𝒊𝒏 = 𝒄𝒄,𝒊𝒏 𝟏 𝟐 = 20 g/L and ∆𝒕𝒄𝒚𝒄 = 5.4 min. (d) The effect of the cycle time on the yield obtained in the single separation process (𝒀𝒄𝟏, hollow square) and the combined separation and racemization process (𝒀𝒕𝒐𝒕 𝟏 , black square). The star on figures a, b and d marks process parameter of the demonstration experiment. Another parameter influencing the productivity is the composition or the enantiomeric excess (ee in %) in the feed tank. In case of incomplete racemization, the remaining enantiomeric excess of the recycled fraction 𝑒𝑒2𝑟 will change the composition of the feed tank in a closed loop arrangement. With each reinjected fraction the composition will gradually shift towards the enantiomer in excess, until a new steady state is reached. The tank composition in the steady state (𝑒𝑒2𝑡𝑎𝑛𝑘 ) is then a function of the ratio between the reintroduced (𝑚1𝑟 , 𝑚𝑠𝑟 ) and injected (𝑚1𝑐,𝑖𝑛 , 𝑚2𝑐,𝑖𝑛 ) masses, which can be expressed by a factor 𝛼 𝑟 24 ACS Paragon Plus Environment

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𝛼𝑟 =

𝑚1𝑟 + 𝑚2𝑟

(31)

𝑚1𝑐,𝑖𝑛 + 𝑚2𝑐,𝑖𝑛

and the corresponding enantiomeric excess of (R)-MA (E2) in the recycled fraction 𝑒𝑒2𝑟 : 𝑒𝑒2𝑟 =

𝑚1𝑟 − 𝑚2𝑟 𝑚1𝑟 + 𝑚2𝑟

(32)

A more concentrated stock solution added periodically in our experiment, used to increase MA concentration in the feed tank, can be also easily considered. The effect of varying 𝑒𝑒2𝑟 for a constant concentration of 20 g/L (S)-MA in the feed tank is depicted in figure 8b. Due to the linear adsorption isotherm of the R-enantiomer, the increase of 𝑒𝑒2𝑟 from 𝑡𝑏 and 1.9% decrease 0% to 10% causes only 0.5% decrease of productivity for the cycle time ∆𝑡𝑐𝑦𝑐 𝑜𝑝𝑡,𝑆

at the optimized cycle time ∆𝑡𝑐𝑦𝑐 . In the presented experimental setup the enzymatic activity in the reactor exceeds the needs of the process as, based on results published in our previous study17, it is capable of racemizing higher concentrations of (R)-MA. Nevertheless this rather minor impact of 𝑒𝑒2𝑟 on the productivity is seen as a beneficial feature of the process in case of possible enzyme deactivation (as mentioned in section 4.2). Moreover if the costs of enzyme production are significant, the amount of racemase used in the reactor can be reduced with only a little decrease of the overall process productivity. The effect of a varying enzyme dosages 𝐷𝑒𝑛𝑧 , reactor volume 𝑉𝑟𝑒𝑎𝑐𝑡𝑜𝑟 , substrate amounts (𝑚1𝑐 , 𝑚2𝑐 ) or enzyme kinetics on the enantiomeric excess of the outlet stream and therefore the productivity, can be summarized as followed:

𝑒𝑒2𝑟 =

𝑐 − 𝑚𝑐 ) ∙ (𝐾 +𝑐 ) (𝑚1 𝑀 12 2

(33)

𝐷𝑒𝑛𝑧 ∙ 𝑣𝑚𝑎𝑥 ∙𝑐12 ∙𝑉𝑟𝑒𝑎𝑐𝑡𝑜𝑟 ∙𝜏

For the adsorption isotherm shape of the studied example, the amount that can be injected on the chromatographic column has a much stronger influence on the process productivity. Figure 8c depicts the effect of injection volume of 40 g/L racemic MA on the purification productivity. In the 25 ACS Paragon Plus Environment


Page 26 of 29

shown range, productivity is almost directly correlated to the injected mass. Doubling the mass 𝑡𝑏 from 2 mg, to 4 mg for a cycle time of ∆𝑡𝑐𝑦𝑐 = 5.4 min, increases productivity by 86.6%. A cycle

time optimization for each injected mass further increases the productivity (data not shown). The sub-optimal injection amount was used in the proof-of-concept experiment due to aforementioned detector limit. In case of larger scale purification, where exact detection of the profiles is not a major goal, this issue would not be crucial and a higher load can be chosen. 𝑡𝑏 Finally, in case of the touching band separation (using ∆𝑡𝑐𝑦𝑐 ) and (S)-MA as a target enantiomer,

the yield of the single chromatographic separation is 49.8% (eq. (30)), compared to 100% for the 𝑜𝑝𝑡,𝑆

combined process including by-product racemization. For the cycle time ∆𝑡𝑐𝑦𝑐 = 3.55 min, the yield of a single separation process 𝑌1𝑐 further decreases to 40.8%, as it is shown in fig. 8d. In comparison, the yield of the combined process 𝑌1𝑡𝑜𝑡 remains at 100%. Thus, the coupled process not only has the advantage of significantly increasing the yield, but also allows to apply shorter cycle times, leading to larger productivities, which can be introduced without affecting the overall yield. 5. CONCLUSIONS AND OUTLOOK A successful example of coupling the batch wise enantioselective chromatography with enzymatic racemization was presented. It has been shown that the two processes can be operated with a common mobile phase. An important novelty was the implementation of a fixed bed reactor with an immobilized enzyme to racemize the unwanted enantiomer. The immobilization improved the lifetime of the enzyme and eliminated the need of using it in excessive amounts. Moreover, ease of biocatalyst re-use further decreases the demand for the enzyme. A fixed bed reactor was found to be a convenient operation unit that can be used in various process configurations. It can be implemented not only for racemization of defined fractions eluting from a single chromatographic column but also for by-product fractions from a multi-column SMB chromatographic unit. The experimental implementation of the process could be improved by an automatization of the recycling step. 26 ACS Paragon Plus Environment

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Process simulations based on a simplified model have been used to find first optimal conditions to provide the desired enantiomer with maximized productivity for a fixed flow rate. It was shown that as long as the racemization in the reactor is complete the process reaches a steady-state quickly (e.g. already in the second cycle). Furthermore, it was shown that the resolution and not the racemization step turned out to be the bottle neck of the presented coupled process The theoretical predictions can be used in the future to further optimize it and to design a process with maximized productivity for the purification of both MA enantiomers. This could be done by overloading the chromatographic column and by adjusting the cycle time. If needed the dimensions of the required larger racemization reactor, with higher total enzymatic activity, could be predicted. To an extend the conversion in the reactor could also be increased by adjustment of the flow rate to match the required residence time in the reactor or by integrating an intermediate tank to compensate the concentration fluctuations at the reactor inlet. It was found, that in case of incomplete racemization the system can be operated with a remaining enantiomeric excess in the recycled fraction without significant loss in productivity. Potential further improvement can be achieved by introduction of a nanofiltration unit to increase the concentration of the fractions exiting the racemization reactor. This would also help to avoid concentration fluctuations in the feed tank. This is beneficial for two reasons. Most importantly, it stabilizes the elution profiles and therefore allows fixed fractionation times, which do not need to be adapted to the changing feed tank concentrations. Secondly, a loss of productivity due to the lower concentrations of the recycled streams and therefore dilution of the feed could be avoided. Altogether, the proposed modelling approach is seen as a powerful tool for optimization of the successfully carried out coupling of chromatography and racemization in a fixed bed reactor. The mandelate racemase reactor presented here could be used as racemization step in separation of several mandelic acid derivatives due to a broad substrate tolerance of mandelate racemase 17,28. The promiscuity of the enzyme makes it an attractive biocatalyst for separation of various nonnatural -hydroxycarboxylic acids increasing the applicability of presented process for industrial application. Moreover high yield separation of other valuable products can be potentially 27 ACS Paragon Plus Environment


designed and optimized with presented approach if a suitable racemase can be found and immobilized. ACKNOWLEDGEMENTS The authors would like to thank Dr. Katja Bettenbrock (MPI, Magdeburg, Germany) for the access to the Experimental Systems Biology laboratories and any helpful suggestions.

The authors declare no financial or commercial conflict of interest. REFERENCES (1)

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Combination of Enantioselective Preparative Chromatography and

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