A combination of HPLC and automated data analysis for monitoring the efficiency of high-pressure homogenization

Abstract

BackgroundCell disruption is a key unit operation to make valuable, intracellular target products accessible for further downstream unit operations. Independent of the applied cell disruption method, each cell disruption process must be evaluated with respect to disruption efficiency and potential product loss. Current state-of-the-art methods, like measuring the total amount of released protein and plating-out assays, are usually time-delayed and involve manual intervention making them error-prone. An automated method to monitor cell disruption efficiency at-line is not available to date.ResultsIn the current study we implemented a methodology, which we had originally developed to monitor E. coli cell integrity during bioreactor cultivations, to automatically monitor and evaluate cell disruption of a recombinant E. coli strain by high-pressure homogenization. We compared our tool with a library of state-of-the-art methods, analyzed the effect of freezing the biomass before high-pressure homogenization and finally investigated this unit operation in more detail by a multivariate approach.ConclusionA combination of HPLC and automated data analysis describes a valuable, novel tool to monitor and evaluate cell disruption processes. Our methodology, which can be used both in upstream (USP) and downstream processing (DSP), describes a valuable tool to evaluate cell disruption processes as it can be implemented at-line, gives results within minutes after sampling and does not need manual intervention.

A Chemometric Tool to Monitor and Predict Cell Viability in Filamentous Fungi Bioprocesses Using UV Chromatogram Fingerprints

“…Samples from twelve (9 SS and 3 LS) P. chrysogenum cultivations were used for acquiring UV chromatographic data through size exclusion chromatography (SEC). UV chromatographic data have been shown to contain process information with respect to nucleic acids (having maximum absorbance at 260 nm) and protein impurities (having maximum absorbance at 280 nm) [23,24]. Significant changes in the impurity release profiles, especially during metabolic stress and viability decline phases, can be used to monitor process performance.…”

“…UV chromatography is one of the most commonly used techniques in various bioanalytical assays. Recently, we employed UV chromatography coupled with chemometric approaches to monitor cell lysis in Escherichia coli bioprocesses [22] and for process development of downstream unit operations [23,24]. In a similar approach, we used principal component analysis (PCA) with UV chromatographic data of samples from twelve P. chrysogenum bioprocesses to monitor cell viability.…”

Prediction of the Quantity and Purity of an Antibody Capture Process in Real Time

“…Actions in DSP have to be placed within seconds, and the measurement has to be suitably fast. At‐line high‐performance liquid chromatography (HPLC) measurements were proposed by different research groups . However, such HPLC measurements require automated sampling and subsequent data analyses to obtain results fast enough to give feedback information.…”

“…At-line highperformance liquid chromatography (HPLC) measurements were proposed by different research groups. [12][13][14][15][16] However, such HPLC measurements require automated sampling and subsequent data analyses to obtain results fast enough to give feedback information. With respect to automated peak cutting, it would be desirable to obtain results within minutes or even seconds.…”

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

“…[2] Most E. coli strains are not able to secrete high amounts of recombinant proteins, therefore the DSP has to include a cell disruption step to access the intracellular product. [2][3][4] Several principles for cell disruption exist, which we recently summarized. [3] In short, chemical, biological, physical and mechanical methods can be used to release the product from the cells.…”