Assessment of the manufacturability of Escherichia coli high cell density fermentations

Abstract

The physical and biological conditions of the host cell obtained at the end of fermentation influences subsequent downstream processing unit operations. The ability to monitor these characteristics is central to the improvement of biopharmaceutical manufacture. In this study, we have used a combination of techniques such as adaptive focus acoustics (AFA) and ultra scale-down (USD) centrifugation that utilize milliliter quantities of sample to obtain an insight into the interaction between cells from the upstream process and initial downstream unit operations. This is achieved primarily through an assessment of cell strength and its impact on large-scale disc stack centrifugation performance, measuring critical attributes such as viscosity and particle size distribution. An Escherichia coli fed-batch fermentation expressing antibody fragments in the periplasm was used as a model system representative of current manufacturing challenges. The weakening of cell strength during cultivation time, detected through increased micronization and viscosity, resulted in a 2.6-fold increase in product release rates from the cell (as measured by AFA) and approximately fourfold decrease in clarification performance (as measured by USD centrifugation). The information obtained allows for informed harvest point decisions accounting for both product leakages during fermentation and potential losses during primary recovery. The clarification performance results were verified at pilot scale. The use of these technologies forms a route to the process understanding needed to tailor the host cell and upstream process to the product and downstream process, critical to the implementation of quality-by-design principles.

Periplasmic expression in and release of Fab fragments from Escherichia coli using stress minimization

“…Since E. coli does not naturally secrete recombinant proteins, this is due to release of periplasmic Fab by outer membrane damage. This is problematic for industrial processes since periplasmic release requires bacteria to be physically intact to permit selective release of periplasmically‐targeted recombinant proteins and prevent release of cytoplasmic proteins during centrifugal cell harvesting or osmotic release procedures . If bacteria are physically weakened, for example by RPP‐induced stress, then far more protein is released, complicating DSP; the advantages of periplasmic targeting and release are minimal compared with cytoplasmic expression, lysis and purification from whole cell lysate.…”

“…We have demonstrated that stress minimization by decreasing culture temperature and inducer concentration can be used to improve not only recombinant protein yields and folding but also correct subcellular targeting and overall cell integrity for subsequent cell harvest and protein release steps . Careful balancing of increases in inducer concentration and decreases in growth and induction temperature allowed optimization of Fab D1.3 yield.…”

“…In order for periplasmic release to succeed, bacterial viability and physical integrity must be maintained upon harvest. Metabolic stress in bacteria generating recombinant proteins such as periplasmically‐targeted Fab fragments can cause cell lysis during fermentation or fragility leading to lysis during cell harvesting in industrial centrifuges or subsequent processing steps . This eliminates the advantages of periplasmic expression as cytoplasmic proteins, membrane components and DNA are all released, complicating subsequent purification of the target recombinant protein.…”

Detecting cell lysis using viscosity monitoring in E. coli fermentation to prevent product loss

“…During fermentation, as the limit of the periplasm is reached, cells begin to lose viability and leak the Fab' product and other intracellular content to the fermentation broth. Apart from product loss in late stage fermentation, the remaining viable cells become more fragile and break fourfold more than those harvested at an earlier stage, if subjected to the equivalent shear level of that in an industrial centrifuge . As cell lysis occurs in late stage fermentation, leakage of product to the fermentation broth also acts to increase the broth viscosity, as large quantities of chromosomal DNA and other intracellular content are released simultaneously into the broth .…”

Maximizing the stability of metabolic engineering‐derived whole‐cell biocatalysts

“…Further, the critical concentration required for effective downstream processing can increase as a function of time in response to the accumulation of contaminants, e.g. arising from cell lysis, in the fermentation broth . The right boundary, determined by the inherent biocatalyst stability, can be tilted inwards, if, for instance, subtoxic product concentrations are accompanied by increased microbial energy demands compromising the efficiency of metabolic network operation .…”