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10.5 Process Development 239

10.5
Process Development
Perhaps the first decision to be made in process development is the difficult decision
of whether the enzymes to be used should be used in an integrated format. Such
a question does not arise with conventional single biocatalytic steps but is highly
important in multienzyme processes. One of the key criteria here is whether the
enzymes can be operated together without compromise to any of the individual
enzyme’s activity or stability. An interaction matrix (see Section 10.6) can be used to
assist such decision making. In cases where the cost of one or more of the enzyme(s)
is not critical, it will be possible to combine in a one-pot operation. In other cases,
where the cost of an individual enzyme becomes critical, then it may be necessary
to separate the catalysts, such that each can operate under optimal conditions.
Likewise, selection of the biocatalyst format (immobilized enzyme, whole cell,
cell-free extract, soluble enzyme, or combinations thereof) in combination with
the basic reactor type (packed bed, stirred tank, or combinations thereof) and
biocatalyst recovery (mesh, microfiltration, ultrafiltration, or combinations thereof)
will determine the structure of the process flowsheet and therefore is an early
consideration in the development of any bioprocess. The criterion for selection of
the final type of biocatalyst and reactor combination is primarily economic and
may best be evaluated by the four metrics in common use to assess the economic
feasibility of biocatalytic processes [29]:

• reaction yield (g product/g substrate);
• biocatalyst yield (g product/g biocatalyst);
• product concentration (g product/l);
• space-time-yield (g product/l/h).

The balance between the four metrics is dependent upon the relative costs
in a process. For example, a process with a high cost of biocatalyst requires a
high biocatalyst yield, whereas those with a high cost of process plant will require
a high space-time-yield and those with a high downstream processing cost require
a high product concentration to leave the reactor.
Following these early decisions about catalyst integration, and selection of the
appropriate biocatalyst and reactor combination, in the case of multienzyme pro-
cesses it is then necessary to embark on a development program to improve
the biocatalyst. One option is to consider the multitude of different forms and
pretreatments of a biocatalyst (e.g., whole-cell options including display, permeabi-
lized, washed permeabilized, intact). A complementary approach is to use genetic
engineering, via expression of the desired enzymes over other proteins. Finally
the third approach is to consider process strategies to overcome limitations and
bottlenecks. In reality, all options should be considered in parallel, but for simplicity
the possibilities of recombinant DNA technology and process engineering will be
considered here.