Cell culture medium improvement by rigorous shuffling of components using media blending

Abstract

A novel high-throughput methodology for the simultaneous optimization of many cell culture media components is presented. The method is based on the media blending approach which has several advantages as it works with ready-to-use media. In particular it allows precise pH and osmolarity adjustments and eliminates the need of concentrated stock solutions, a frequent source of serious solubility issues. In addition, media blending easily generates a large number of new compositions providing a remarkable screening tool. However, media blending designs usually do not provide information on distinct factors or components that are causing the desired improvements. This paper addresses this last point by considering the concentration of individual medium components to fix the experimental design and for the interpretation of the results. The extended blending strategy was used to reshuffle the 20 amino acids in one round of experiments. A small set of 10 media was specifically designed to generate a large number of mixtures. 192 mixtures were then prepared by media blending and tested on a recombinant CHO cell line expressing a monoclonal antibody. A wide range of performances (titers and viable cell density) was achieved from the different mixtures with top titers significantly above our previous results seen with this cell line. In addition, information about major effects of key amino acids on cell densities and titers could be extracted from the experimental results. This demonstrates that the extended blending approach is a powerful experimental tool which allows systematic and simultaneous reshuffling of multiple medium components.

Chinese hamster ovary cell performance enhanced by a rational divide-and-conquer strategy for chemically defined medium development

“…Medium optimization of each cell line in our current study was completed within 8 weeks with fewer than 60 tests with 1.5e3-fold improvement in the observed product yield. This strategy was demonstrated to be efficient, effective, scalable and universal in four different cell lines, and it showed a significant advantage over other strategies (4,12).…”

Tailoring recombinant protein quality by rational media design

“…Cell culture media design has a great potential to modulate the key quality attributes of the molecule 25 , and in particular by means of high-throughput design of experiment (DoE) approaches 26,27 , due to its central role in the upstream manufacturing 19 . The environment the cell is cultured in closely influences its metabolism to a great extent [20][21][22] .…”

“…The adjustment of the levels of a choice of medium components and the addition of novel compounds either in the medium or the in bolus feeds, influence the enzymatic reactions in the endoplasmic reticulum and in the Golgi apparatus, the substrate generation as well as their transport into the two organelles 23,24 . Further enhancements and technology advancements of high-throughput cell culture systems, including 96-deepwell plates and TubeSpin bioreactor tubes, allow to test a plethora of different supplements at various concentrations simultaneously [25][26][27] . Moreover, the observed trends in these systems may be reproduced in controlled bioreactor systems, including lab-scale bioreactors 28 , which highlights the great value of small-scale high-throughput compound screening for the identification of new levers that tailor recombinant protein quality attributes.…”

“…This chapter presents potential compounds and mechanisms influencing the production of low-molecular-species. High-throughput media blending approaches 26,27,251 and mechanistic studies were called upon based on the information provided by the literature and hypotheses resulting from the experimental results. Due to the relatively few number of scientific reports that address LMW formation in mammalian cell culture, it was decided to perform a general reshuffling of the platform medium components to identify potential levers for LMW content reduction.…”

“…High-throughput cell culture systems opened the door for testing many conditions simultaneously [266][267][268] . The technology developed for media optimization in 96-deepwell plates (96-DWP) fed-batch cultures 26,27,251 can also be applied for product quality modulation 25,64 . Good alignment of the glycan profiles between 96-DWP and shake tubes has been shown, and in the same study trends observed in high-throughput systems have been confirmed in lab-scale bioreactors 28 .…”