Choosing the proper enzyme and optimizing its function:
Within the six major classes of biocatalytic enzymes, there are many natural and now non-natural or recombinant enzyme choices. Just as with any small molecule reaction, an optimal 'stoichiometry' of reagents-to-enzyme catalyst can be determined by DoE practices, automated by synthesis reactors and aided by PAT integrated in process development. Enzymes are biologically evolved to be relatively unstable and amenable to breakdown and recycle. Understanding the practical half-life and stability of any free-, substrate-bound or surface-immobilized enzyme can be critical to the design of proper biocatalysis protocols.
Buffers and Solvents:
Many natural enzymes struggle to cope with organic solvent reactions. A constantly evolving profile of engineered or recombinant enzymes are improving compatibility with organic solvent conditions. Such conditions may not only be amenable, but also advantageous for the reaction efficiency of several small molecule products and by-products and are employed today with greater frequency. Although, use of organic solvents is still not common when the product or by-product of a biocatalytic transformation results in a more traditional biologic molecule such as peptide or antibody conjugates.
In the past, industrial oligonucleotide synthesis has struggled to employ biocatalysis due to the need for modified phosphoramidite monomers which are not compatible with natural polymerases and other DNA/RNA replicating enzymes. The use of some organic solvent conditions are also starting to be explored along with an emergence of industrial suited recombinant enzymes which are capable of catalyzing reactions in organic solvent environments and/or utilizing modified phosphoramidites in oligonucleotide synthesis.
Process parameters – pH:
Natural or recombinant enzymes are still biological molecules governed in their own structure and functions through extensive intramolecular electrostatic interactions and also affected by the presence of co-factors, co-enzymes, or other allosteric effectors. It is well known that enzymes generally operate best within narrow pH ranges and tolerate pH variation <0.5 pH units. Thus, pH control is often a critical parameter for consideration for many biocatalytic reactions and may be difficult or tedious to control manually. The product or by-products of a biocatalytic reaction can also be affected by the control of reagent stoichiometry and other free acids or bases, each of which is driven by a balance of their collective, net pKa; too far outside of an ideal operational space and the reactions may be adversely effected.
Process parameters – Temperature:
Natural enzymes, and the recombinant counterparts engineered from them, have evolved to function efficiently at specific temperatures with fairly tight ranges. Temperature may not always be difficult to control but it is always important to get correct. Deviations may cause changes in reaction kinetics, product yield or other critical process outcomes.
Process parameters – Dissolved Oxygen:
Especially important for oxygenation and reduction reactions, this critical process parameter is usually controlled by sparge or headspace dosing of oxygen or inert gas. It then becomes critical to optimize the quantity and mass transfer (or kLA) of dissolved oxygen into the reaction mixture to ensure that oxidoreductase enzymes can operate at peak efficiency and mitigates risks of unwanted by-product formation.
Process parameters – mixing, fluid dynamics and gases
On the surface a 'well mixed reaction' ensures homogeneity and even reaction kinetics. But mixing also hides potential pitfalls which can easily be overlooked. The use of a proper mixing apparatus can have a great affect on the life cycle of biocatalytic enzymes, especially over long duration or flow chemistry or perfusion style runs. Scale-up from early process development may be especially susceptible if mixing is insufficiently characterized; likely to utilize a simple glass beaker set up on a stir plate with magnetic stir bar or another high sheer method of mixing and may result in poor reaction kinetics but may also be a cause of time-dependent reaction efficiency decreases.
Mixing is also critical for another parameter already addressed, gas distribution and mass transfer. Combined with optimization of the gas dosing/ sparging method, mixing conditions significantly affect the concentration and absorption of gases into the biocatalytic reaction.