Pitfalls in Early Bioprocess Development Using Shake Flask Cultivations

Abstract

For about 100 years, the shake flask has been established for biotechnological cultivations as one of the most important cultivation systems in early process development. Its appeal lies in its simple handling and highly versatile application for a wide range of cell types-from bacteria to mammalian cells. In recent decades, extensive research has been conducted on the shake flask, to not perform processes blindly but to gain a deeper understanding of the various process parameters, phenomena, and their impact on the process. Although the characterization of the shake flask is now well-established in literature, many publications show that this knowledge is often inadequately applied. Therefore, this review provides an overview of the current state of knowledge on various topics related to the shake flask. We first present the key process parameters and their influence on different physical phenomena, such as power input, the largely unknown in-phase/out-of-phase phenomenon, as well as temperature and mass transfer. Then, the most common online monitoring systems that have been established for shake flasks are discussed. Finally, various pitfalls that often arise from inadequate knowledge of handling shake flask cultivations are discussed and guidance on how to avoid them is provided.

Keywords: biochemical engineering; bioprocess development; cultivation system; online monitoring; shake flask.

FIGURE 1

Development of number of publications per year on commonly used systems for biotechnological cultivations between 1900 and 2023. Google Scholar searched for “shake flask”, “microtiter plate”, and “stirred tank reactor”. The search was made on Jul 16, 2024, using https://scholar.google.com/. For clarity, only every fourth data point is plotted as a symbol. Lines are drawn through all data points.

FIGURE 2

Setup of a shake flask cultivation. Process parameters—liquid filling volume, maximum inner flask diameter, shaking frequency, and shaking diameter—are written in red.

FIGURE 3

Cultivation parameters provided in publications on shake flask fermentations with Escherichia coli and thereof calculated risk of oxygen limitation. Scopus database searched for “scherichia coli” and “production” in “Chemical engineering”. The search was made on July 1, 2024, using https://www.scopus.com/. Only research conducted in non‐baffled shake flasks and with the goal of aerobic production was considered for evaluation. Cultivations for genetic engineering purposes were not considered. The most recent 15 articles matching all criteria were analyzed. The OTR_max was calculated using Equation (21) [25]. If not explicitly provided in the publication, a shaking frequency of 250 rpm and a shaking diameter of 25 mm were assumed for calculation. In cases where information on the shake flask dimensions or liquid filling volume was missing, the OTR_max could not be calculated. The risk of oxygen limitation was categorized as low (>20 mmol∙L⁻¹∙h⁻¹), medium (15–20 mmol∙L⁻¹∙h⁻¹), and high (<15 mmol∙L⁻¹∙h⁻¹) based on the OTR_max [26, 87].