Microscale disruption of microorganisms for parallelized process development

Abstract

Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris are the standard platforms for biopharmaceutical production with 40% of all between 2010 to 2014 approved protein drugs produced in those microbial hosts. Typically, products overexpressed E. coli and S. cerevisiae remain in the cytosol or are secreted into the periplasm. Consequently, efficient cell disruption is essential for high product recovery during microbial production. Process development platforms at microscale are essential to shorten time to market. While high-pressure homogenization is the industry standard for cell disruption at large scale this method is not practicable for experiments in microscale. This review describes microscale methods for cell disruption at scales as low as 200 µL. Strategies for automation, parallelization and miniaturization, as well as comparability of the results at this scale to high pressure homogenization are considered as those criteria decide which methods are most suited for scale down. Those aspects are discussed in detail for protein overexpression in E. coli and yeast but also the relevance for alternative products and host such as microalgae are taken into account. The authors conclude that bead milling is the best comparable microscale method to large scale high-pressure homogenization and therefore the most suitable technique for automated process development of microbial hosts with the exception of pDNA production.

Effective Release of Intracellular Enzymes by Permeating the Cell Membrane with Hydrophobic Deep Eutectic Solvents

“…Because mechanical methods are nonspecific, with release of the entire intracellular content, which increases the difficulty of further processing, some nonmechanical methods have been conducted, such as physical disruption, chemical permeabilization, and enzymatic disruption . Chemical permeabilization could be attained by chelating agents, chaotropes, detergents, or organic solvents .…”

“…The comparison was conducted under the same conditions, except for the concentrations of chemical penetrants. The traditional chemical penetrants were used at the optimal concentrations reported previously . The results are shown in Figure .…”

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

“…Various other HT cell disruption/lysis devices have been used, including an 8well-sonifier for VLPs from E. coli, a 24-well-HT sonication device for 15 cells including bacteria, fungi, and yeasts, and microfluidic channels (96-well-format) for thermal treatment, osmotic shocks, and freeze-drying (Baumann and Hubbuch, 2017). In a review, microscale disruption of microorganisms (as low as 200 µl) for parallelized process development was discussed in detail along with their performance compared with high-pressure homogenization (Walther and Dürauer, 2017). HT refolding systems for IB-expressed proteins are also available commercially and are listed in Table 5 (Baumann and Hubbuch, 2017), together with some of the other HTP devices used in downstream process development.…”

An ultrasonic biosample disruptor with two triangular teeth on its radiation face

“…[2,3] Ultrasonication could disrupt the algal cells effectively. [4,5] Compared with other methods, it involves no assistance materials, and its energy requirement is relatively low. [6] Ultrasonication could also be used to disrupt algal cells, in a combination with other methods such as H 2 O 2 oxidation, alkali, nozzle spraying, high-pressure homogenization.…”