Escalamiento

Optimizing scale-up fermentation processes
Michel Thiry and Doriano Cingolani

Opinion

TRENDS in Biotechnology Vol.20 No.3 March 2002

103

There are many aims associated with the optimization of fermentation processes. Optimization is expected to increase the yield of the final product but the process must be compliant with good manufacturing practices, the available equipment and theexpected final scale of operation. Dealing with genetically modified microorganisms that overproduce recombinant protein has the advantage that the vast majority of the processes use only three different species, namely Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris. Standard processes for each organism are described in textbooks and serve as a basis for the development of atailored process. This article outlines the general philosophy that we have devised to ensure an efficient approach of scaling up fermentation processes for biopharmaceutical purposes, in a multidisciplinary environment.
The optimization of the fermentation process is included in the strategic analysis of the viability of a project. Optimization takes place once the feasibility of the production inthe selected organism has been demonstrated. This implies that an expression system has been constructed and that, at least from a theoretical viewpoint, the system should be regarded as the optimum. Before starting long and expensive optimization work, it is important that the stability of the strain is established, at least for the number of generations necessary for cell banking andlargest-scale fermentation, including the pre-cultures. Expression systems based on plasmids are sometimes unstable – several parameters affect the segregational stability of plasmids. The stability is different for each plasmid and depends on the host strain [1]; high instabilities are correlated with low copy number plasmids [2]. The size of the inserted DNA affects the stability; the larger the plasmidis, the less stable it is [3]. Culture conditions, such as temperature [4], the composition of the media and the growth rate [5], can also modify the plasmid stability. Plasmid loss is significant over the postexponential growth phase [6] and plasmid instability is enhanced during the period of gene expression. Antibiotics are often used to stabilize plasmids. The compensation of an auxotrophicdefect of the host coded on the plasmid [7] is more suitable than using antibiotics for regulatory reasons. However, the preparation of media for auxotrophic strains is laborious. The plasmid can be stabilized by the insertion of the parB (hok/sok) locus, which kills the plasmid-free cells by segregation [8]. An alternative is the integration of the insert in the host’s

chromosome. This isoften the case for Pichia pastoris constructions but gene integration can reduce the expression level [9]. The choice of the producing strain and the expression vector will depend on the constitutive or inducible expression into the cytoplasmic compartment or the secretion into the outer medium. If the strain has been acquired outside the laboratory, the quality of the documentation of the source ofthe original strain and the cloning procedure must be evaluated and approved by a competent quality assurance service. The codon usage of the gene of interest should be optimized to facilitate the expression in the chosen microorganism. A screening of expression in a shake-flask must be performed immediately to find a clone that produces the recombinant protein at a high level of expression.Optimization of culture conditions
Given that the main aim of optimization is to maximize the production, this process can be initiated only once a laboratory-scale purification process and a minimum set of quality control tools are available to quantify and assess the quality of the product. The fermentation protocol affects the impurity profile and thus impacts strongly on the efficacy of the...