Shake tube perfusion cell cultures are suitable tools for the prediction of limiting substrate, CSPR, bleeding strategy, growth and productivity behavior

Abstract

BACKGROUND Scale‐down models (SDM) are crucial for efficient setting of different experimental conditions in cell culture media‐ or clone‐screening applications and to predict the expected performance in larger bioreactor cultivations. However, the availability of such models for continuous bioprocessing are scarce and hampered by the requirement for proper cell retention in perfusion applications. RESULTS In this study, perfusion scale‐down models were optimized for increased cell performance in shake tubes by a semi‐continuous operation mode without any instrumental monitoring. A 40 L perfusion bioreactor was simulated with 10 mL shake tubes by daily medium exchange with or without cell bleeding for process fine‐tuning. Specifically, the optimal cell‐specific perfusion rate (CSPR) was efficiently identified using the perfusion SDM. The cellular growth and productivity were nearly identical to those of the large‐scale experiments. The 24‐h interval of metabolite analysis identified the limits of the SDMs and underscores the need for perfusion processes in large‐scale animal cell bioprocesses. CONCLUSION Optimized perfusion scale‐down models in semi‐continuous operation mode are highly suitable tools to define important process parameters of larger perfusion bioreactor cultures and are predictive of cellular growth and production behavior.

Combined gene and environmental engineering offers a synergetic strategy to enhance r‐protein production in Chinese hamster ovary cells

“…Testing this hypothesis will require the use of bioreactors with a controlled environment, so we can discard the contribution of other environmental factors, such as pH, osmolality, or oxygen supply. Although spin tubes are effective scaledown models for evaluating cell performance and setting parameters in perfusion systems (Mayrhofer et al, 2021;Wolf et al, 2019Wolf et al, , 2018, it remains to be further evaluated whether the product yields and cell-specific productivities reached in our CHOBIgG1 cells treated with NaBu can be translated into large-scale continuous perfusion processes.…”

“…Semi-continuous perfusion cultures were performed using CHO IgG1 , CHOB IgG1 , CHO EPOFc, and CHOB EPOFc cell lines by replacing medium daily as previously described (Mayrhofer et al, 2021;Wolf et al, 2019Wolf et al, , 2018. Cells were seeded at 1 × 10 7 cells/ml in 10 ml growth medium supplemented with 10% (vol/vol) CHO CD EfficientFeed™ A (Gibco) and 10% (vol/vol) CHO CD EfficientFeed™ B (Gibco).…”

“…To address this point, we evaluated whether a strategy of combined BLIMP1 overexpression and NaBu treatment generated higher product yields in high-density cultures. For this, we performed semicontinuous perfusion cultures in shake tubes by centrifugation and resuspension of cells in a fresh medium every 24 h. The use of shake tubes represents a cost-effective approach for performing perfusion cultures, with comparable results as a scale-down model compared to controlled bioreactors in CHO cells (Mayrhofer et al, 2021;Torres, Elvin, et al, 2021;Wolf et al, 2019Wolf et al, , 2018. For these studies, we used recombinant CHO cell lines that stably expressed BLIMP1 (referred to as CHOB IgG1 and CHOB EPOFc ) derived as described in our previous paper .…”

Optimization of medium with perfusion microbioreactors for high density CHO cell cultures at very low renewal rate aided by design of experiments

“…The complexity of a perfusion bioreactor with a cell retention system and feed, harvest and cell bleed lines, is challenging to obtain in a parallel format in milliliter‐scale systems. Although not being a continuous system, the spin tube operated in semi‐perfusion mode is a model that has been extensively used to mimic perfusion bioreactors (Gomez et al, 2017; Mayrhofer et al, 2021; Villiger‐Oberbek et al, 2015). Such a model can be a supportive tool to assist process development with resource‐intensive perfusion bioreactors.…”

Process intensification for CHO cell biomanufacturing

“…However, just a slightly lower CSPR, e. g. 40 pL•(c•d) -1 , might have been sufficient as well, thus saving 20 %. Nowadays, CSPRs as low as 10 to 20 pL•(c•d) -1 are being reported 194 using design of experiment studies 195,196 , whereas the exact composition is naturally specific and confidential. As the CSPR is in essence just one universal parameter comprising all media components, targeted feeding of specific components 27,197,198 can be applied if i) known to become critical, ii) they can be ideally measured on-line, e. g. using Raman spectroscopy (see chapter 6.3.3) and iii) a functional control architecture is set-up to control feed rates.…”