Marco conceptual

1- based on enzyme saturation for NeuSAc resolution (Uchida et al, 1984, Ohta et a l, 1988, Aisaka et a l, 1991,

Ch a p t e r 3: Pr o c e s s Op t io n Ev a l u a t io n

Table 3.2 Processing bases for reactor selection and operation.

Limit Criterion for Reactor Evaluation

enzyme productivity' / unit enzyme

operational stability

DSP product concentration

product / contaminants substrate and product conversion

productivity* / unit volume

time productivity*

operation time

Ch a p t e r 3: Pr o c e s s Op t io n Ev a l u a t io n

product costs. Eventually, minimisation o f timing would reduce operating expenditures o f production units.

The following designs have been selected for further evaluation under possible reaction conditions (Chapter 5):

• Biotransformation with pyruvate feed

• Biotransformation with feed o f ManNAc and Pyr to maintain Pyr below 200 mM and M anNAc below 750 mM.

• Biotransformation in a plug flow reactor. • Biotransformation with in NeuSAc removal.

Batch reaction with excess Pyr has been already assessed (Chapter 2). Feeding strategies would be beneficial for enzyme kinetics and possible reactor-downstream integration (Section 3.4). A plug flow reactor can be operated in continuous mode with the benefits o f a batch kinetics in relation to NeuSAc inhibition. In addition. In situ product removal would overcome NeuSAc detrimental effects together with equilibrium constraints and achieve full substrates stoichiometric conversion. The validation o f these reactor configurations and their operating characteristics will be investigated in Chapter S for different processing frameworks.

3.4 INTEGRATED PROCESSES

In the design o f bioprocesses, the potential o f a step change in production techniques could overcome the constraints imposed on a particular biotransformation and permit processes otherwise unfeasible (W oodley and Lilly, 1996). Moreover, the integration o f production with other steps o f the process is required in order to select optimal flow sheet (Cooney et a l, 1988; Bruin, 1992) and operating strategies (Ingleby, 1986; M iddleberg et a l, 1992). There are several process advantages when the production step is integrated with downstream processing operations. Minimisation o f interferences due to product accumulation, reduction o f product losses due to the reaction conditions and limitation o f

C h a p t e r 3: Pr o c e s s Op t io n Ev a l u a t io n

biotransformation (Freeman el al. 1993). Moreover, an unfavourable thermodynamic equilibrium may be shifted by in situ product removal (Woodley and Lilly, 1994).

The characteristics o f NeuSAc biotransformation have been investigated to determine the process options and in particular the feasibility o f integration o f a biotransformation with either the upstream chemical épimérisation or the downstream product recovery. One o f the sim plest techniques for recovery o f the product, NeuSAc, is crystallisation in acetic acid at low pH (Lin et a l, 1992). Although all the reactants and the product were found to be relatively stable at pH 2 (Table 2.2) the enzyme loses activity below pH 3.S (Uchida et a l,

1984) thus there is no justification for integration o f the biotransformation and product recovery, unless an alternative to crystallisation is used. However, other methods o f in situ

product removal (Freeman et a l, 1993), such as ion exchange chromatography (Schauer, 1973), may prove possible and would improve the conversion yield and reaction rate. The evaluation o f an in situ product removal system for the synthesis o f NeuSAc will be developed in Chapter 5.

The advantage o f integration between the chemical épimérisation and the biotransformation step is that it overcomes the unfavourable equilibrium for conversion o f GlcNAc to M anNAc by removal o f the latter compound by the biotransformation. The rate o f épimérisation o f GlcNAc to ManNAc is highly pH-dependent (Lee, 1990) and operation at pH 10.5-11 is necessary for a reasonable conversion rate. Preliminary experiments to determine the characteristics o f the biotransformation showed that at high pH pyruvate was particularly unstable itself (Table 2.2), although it is reported to have a protecting role on NeuSAc aldolase deactivation (Uchida et a l, 1984). Batch biotransformations at pH 9-11 have been reported (Tsukada and Ohta, 1994) but the results reported in Chapter 2 also indicate an inevitable loss o f pyruvate through alkaline degradation. Thus selection o f the appropriate operating conditions for the combined épimérisation and biotransformation requires further investigation (Chapter 4).

C h a p t e r 3: Pr o c e s s Op t io n Ev a l u a t io n

3.5 SUM M ARY

In this chapter, the characterisation o f the chemo-enzymatic synthesis o f Neu5Ac has provided constraints as a basis for a rational approach to process selection, identifying operating limits at an early stage and ruling out potential integrated process options. Pyruvate was found to be inhibitory at high concentrations, suggesting the need for feeding. As a result o f this approach, an operating window was defined, relating the process boundaries in the form o f a diagram. Feeding strategies for both pyruvate and M anNAc together with plug flow reactor configuration have been selected for further evaluation to the imposed process constraints

(Chapter 5). The rationale o f integration between the biotransformation step together with either the épimérisation (Chapter 4) and ion exchange chromatography (Chapter 5) is discussed.

Ch a p t e r 4: Ep im é r is a t io n - Bio t r a n s f o r m a t io n In t e g r a t io n

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