Development of a high‐throughput microscale cell disruption platform for Pichia pastoris in rapid bioprocess design

Abstract

The time and cost benefits of miniaturized fermentation platforms can only be gained by employing complementary techniques facilitating high-throughput at small sample volumes. Microbial cell disruption is a major bottleneck in experimental throughput and is often restricted to large processing volumes. Moreover, for rigid yeast species, such as Pichia pastoris, no effective high-throughput disruption methods exist. The development of an automated, miniaturized, high-throughput, noncontact, scalable platform based on adaptive focused acoustics (AFA) to disrupt P. pastoris and recover intracellular heterologous protein is described. Augmented modes of AFA were established by investigating vessel designs and a novel enzymatic pretreatment step. Three different modes of AFA were studied and compared to the performance high-pressure homogenization. For each of these modes of cell disruption, response models were developed to account for five different performance criteria. Using multiple responses not only demonstrated that different operating parameters are required for different response optima, with highest product purity requiring suboptimal values for other criteria, but also allowed for AFA-based methods to mimic large-scale homogenization processes. These results demonstrate that AFA-mediated cell disruption can be used for a wide range of applications including buffer development, strain selection, fermentation process development, and whole bioprocess integration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:130-140, 2018.

A Biological OR(XNOR) Logic Gate Couples Carbon Source and Transgene Expression Switching in a Komagataella phaffii (Pichia pastoris) Strain Co-producing Process-Enhancing Lipase and a Virus-like Particle (VLP) Vaccine

“…Western Blotting of Samples from Standard Shake Flask Cultivation. Four milliliters of K. phaff ii inoculum from standard shake flask cultivation was centrifuged at 10000g for 5 min, supernatant was removed, and cell pellet was resuspended in 0.5 mL of the disruption buffer 19 consisting of 50 mM 3-(Nmorpholino)propanesulfonic acid (MOPS), 1 mM 4-benzenesulfonyl fluoride hydrochloride, 5 mM dithiothreitol, 5 mM ethylenediaminetetraacetic acid, and 0.1% (v/v) Triton X-100, adjusted to pH 7.5 using 1 M sodium hydroxide. The resulting cell solution was disrupted using a SoniPrep sonicator with 6 s on and 10 s off for 3 cycles with 10000× amplitude.…”

Bacterial Lysates as Immunotherapies for Respiratory Infections: Methods of Preparation

“…They have developed focused-ultrasonication, an advanced computer-controlled technology that can control the dosage of energy delivered to gently disrupt the cell membranes of mammalian cells or abruptly disrupt the cell walls of bacteria. This technology could address the challenges of standardizing procedures for producing mechanical bacterial lysates (Wenger et al, 2008;Bláha et al, 2017).…”