Demonstrating Scalable T-Cell Expansion in Stirred-Tank Bioreactors
Alex Klarer | July 5, 2018
Cell therapy products have started to gain significant traction in the pharmaceutical industry in the last few years because of the potential they possess to treat many life-threatening conditions. Chimeric antigen receptor (CAR) T-cell therapies have been particularly prominent since the U.S. Food and Drug Administration (FDA) approved Novartis’ Kymriah and Gilead’s Yescarta treatments in late 2017.
However, as promising as cell therapy is for the treatment of cancer and other conditions, there are still considerable challenges to overcome for cell therapy developers if they’re going to make their products commercially viable. One particular challenge is the need to establish a cell culture expansion method that consistently produces a significant yield of viable cells.
Many cell culture methods have a large footprint in cleanroom environments, which minimizes the number of simultaneous operations that can occur per square foot of cleanroom available. This increases costs and reduces production capacity.
To investigate possible methods of improving cell culture expansion, I participated in an experiment alongside David Smith, Ryan Cassidy, Thomas Heathman, and Qasim Rafiq. In this study, we used an ambr 15 microbioreactor to establish whether or not it would be possible in stirred-tank bioreactors.
The ambr 15 microbioreactor was used in this study as it is established as an effective and representative process development system for small-scale and cost-effective culture analysis. With two chambers that can each hold 12 reactors, the ambr 15 microbioreactor allowed for 24 concurrent cell growth tests—saving time and space. Additionally, the operation of the microbioreactor was largely automated, minimizing the risk of variability from human interaction.
This microbioreactor system is invaluable for rapid iteration of small cell culture samples that match the kinetics and forces present in full-scale bioreactor cultures and test the viability of different culture methods.
Here are a few highlights from the study, which you can find the full text of in the June 2018 issue of BioProcess International:
The study had two primary goals:
1. To demonstrate that the proliferation of T-cells in stirred-tank bioreactors is possible—which includes establishing the key conditions and expected cell density in a target culture system without perfusion.
2. To optimize bioreactor growth conditions to establish that the ambr 15 system offers a favorable comparison as a development platform for T-cells with testing for batch-fed and static cultures.
Cell Growth Was Typically Higher with Higher Perfusion Rates
In testing the effects of perfusion—the removal of media from the culture tank and replacing it with fresh media—it was noted that in the majority of cases, higher perfusion rates led to increased cell growth.
During the study, three cell culture samples from three unique donors were put through four different growth processes:
An ambr bioreactor process with low perfusion rates,
An ambr bioreactor process with high perfusion rates,
A batch-fed process, and
A static control process.
In two of the three donor batches, the high-perfusion ambr growth method had significantly higher cell growth rates and final cell counts than the other three methods at the end of 12 days.
While average growth rates for the perfusion processes was initially negative for the first few days, the growth rate for these processes rapidly increased after day four. Meanwhile, the growth rate of the static cell cultures peaked at day four, and then regressed from there. The batch-fed cultures all experienced relatively steady growth over the full 12-day period.
Only one high-perfusion culture batch underperformed in terms of growth compared to the other methods—an anomaly that does not have a clear cause indicated in the data.
Lactate Buildup and Cell Growth
The growth rate data for the cells leaves us with two questions:
1. Why was the period between days five and seven the exponential growth phase for the perfusion processed batches? And;
2. Why did growth stagnate after day eight?
Part of the cause may be the buildup of lactic acid which slows cell growth. At the eight-day mark, the concentration of lactate—a byproduct of the anaerobic energy production process—began to increase, inhibiting the growth of new cells in all four cell culture methods. The removal of the growth media in the perfusion-based processes helped to remove some of the excess lactate, which enabled further growth compared to the other culture methods. However, at the eight-day mark, the lactate began to build up faster than it could be removed through perfusion—slowing cell growth.
This hypothesis is corroborated by an examination of the lactate levels in each tank of the bioreactor. As lactate levels increased, cell growth would plateau. In the study, lactate concentrations would approach a concentration of about 7.6 milligrams per milliliter (mg/mL). In the low-perfusion tanks where 25% of the growth media would be exchanged per day, lactate levels would approach the maximum concentration. The high-perfusion tanks, on the other hand, stabilized their lactate concentration at 7.19 mg/mL. However, this lower concentration was still enough to cause cell growth to stagnate.
In short, the data show a correlation between sustaining a concentration of lactate dehydrogenase above 7.0 mg/mL and cell death.
The Effect of Donor Variability on Cell Growth
In the stirred-tank microbioreactors that underwent high perfusion, two out of the three donor samples saw marked increases in overall cell growth versus the static and batch-fed samples from the same donors. However, one donor saw markedly worse growth with the perfusion process.
This highlights a potential impact of donor variability on the efficacy of the perfusion method for cell cultures. However, to fully establish these effects, further testing will be necessary—preferably with more robust analysis of contaminants (CD14, CD19, CD56) and T-cell exhaustion both pre- and post-culture to gain a better understanding of the variation between donors.
Overall, the experiment demonstrated the viability of stirred-tank bioreactors for cell culture growth, as well as the value of microbioreactors for rapidly iterating different cell culture methods.
Furthering knowledge of T-cell expansion through experiments such as this is important for developing more robust manufacturing protocols for future cell therapy products. To learn more about the experiment, you can find the full report in the June 2018 issue of BioProcess International, or download the PDF of the report at the link below:
 Nienow AW, et al. The Physical Characterisation of a Microscale Parallel Bioreactor Platform with an Industrial CHO Cell Line Expressing an IgG4. Biochem. Engin. J. 76, 2013: 25-36; doi10.1016/j.bej.2013.04011.
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