Overall Key Performance Indicator to Optimizing Operation of High-Pressure Homogenizers for a Reliable Quantification of Intracellular Components in Pichia pastoris

Abstract

The most commonly used cell disruption procedures may present lack of reproducibility, which introduces significant errors in the quantification of intracellular components. In this work, an approach consisting in the definition of an overall key performance indicator (KPI) was implemented for a lab scale high-pressure homogenizer (HPH) in order to determine the disruption settings that allow the reliable quantification of a wide sort of intracellular components. This innovative KPI was based on the combination of three independent reporting indicators: decrease of absorbance, release of total protein, and release of alkaline phosphatase activity. The yeast Pichia pastoris growing on methanol was selected as model microorganism due to it presents an important widening of the cell wall needing more severe methods and operating conditions than Escherichia coli and Saccharomyces cerevisiae. From the outcome of the reporting indicators, the cell disruption efficiency achieved using HPH was about fourfold higher than other lab standard cell disruption methodologies, such bead milling cell permeabilization. This approach was also applied to a pilot plant scale HPH validating the methodology in a scale-up of the disruption process. This innovative non-complex approach developed to evaluate the efficacy of a disruption procedure or equipment can be easily applied to optimize the most common disruption processes, in order to reach not only reliable quantification but also recovery of intracellular components from cell factories of interest.

A step forward to improve recombinant protein production in Pichia pastoris: From specific growth rate effect on protein secretion to carbon-starving conditions as advanced strategy

“…To study the effect of the secretory pathway saturation on the bioprocess efficiency, it is of capital interest the reliable quantification and recovery of the total amount of product accumulated intracellularly along a cultivation [20,21]. Some previous studies focused on the efficacy of high-pressure homogenization disruption procedures on methanol-based cultivations, as it is known that cells growing on this substrate present a significant widening of the cell wall thickness [22,23]. In addition, since an important amount of the protein of interest is expected to be retained through the secretory pathway, besides the soluble part of the cell lysates, the insoluble fraction must be taken into account in order to avoid an underestimation of the target product, as it contains the cell membranes, endoplasmic reticulum (ER), Golgi and other organelles where the protein of interest may be retained [24].…”

Autolysis of Pichia pastoris induced by cold

“…Accordingly, reducing the cost of the extraction and purification is of great economic importance. Cells have to be lysed often by drastic methods to recover intracellular proteins and this can affect the product and thus the yield (Garcia-Ortega et al 2015 ). A major impediment to reduce production costs of recombinant proteins is the availability of a low-cost recovery technology that does not affect adversely the protein and/or the environment (Balasundaram et al 2009 ).…”

A fast and simple approach to optimize the unit operation high pressure homogenization - a case study for a soluble therapeutic protein inE. coli

“…HPH presents an efficient and scalable method for bacterial cell disruption, but has also been applied for other organisms. [18][19][20] In the literature, several studies exist, in which its efficiency has been analyzed, but only regarding cell disruption (cellular integrity and viability) or based on product titer. Within this study, we show how different parameters can influence not only lysis efficiency and product titer during HPH, but also product related properties such as product oligomer purity and charge variance of a soluble ThP.…”