Demonstrating the Manufacture of Human CAR‐T Cells in an Automated Stirred‐Tank Bioreactor

Abstract

Chimeric antigen receptor T‐cell (CAR‐T) therapies have proven clinical efficacy for the treatment of hematological malignancies. However, CAR‐T cell therapies are prohibitively expensive to manufacture. The authors demonstrate the manufacture of human CAR‐T cells from multiple donors in an automated stirred‐tank bioreactor. The authors successfully produced functional human CAR‐T cells from multiple donors under dynamic conditions in a stirred‐tank bioreactor, resulting in overall cell yields which were significantly better than in static T‐flask culture. At agitation speeds of 200 rpm and greater (up to 500 rpm), the CAR‐T cells are able to proliferate effectively, reaching viable cell densities of >5 × 106 cells ml‐1 over 7 days. This is comparable with current expansion systems and significantly better than static expansion platforms (T‐flasks and gas‐permeable culture bags). Importantly, engineered T‐cells post‐expansion retained expression of the CAR gene and retained their cytolytic function even when grown at the highest agitation intensity. This proves that power inputs used in this study do not affect cell efficacy to target and kill the leukemia cells. This is the first demonstration of human CAR‐T cell manufacture in stirred‐tank bioreactors and the findings present significant implications and opportunities for larger‐scale allogeneic CAR‐T production.

The Impact of Fluid Dynamic Stress in Stirred Bioreactors – The Scale of the Biological Entity: A Personal View

“…However, the present approaches impact on consistency of production and cell quality as well as being very expensive and the establishment of scalable, controlled stirred systems should lead to a much better and cheaper process. The work summarized here is a significant move towards that goal [33,49].…”

“…This finding reinforced the concept that once agitation was intense enough to ensure adequate suspension of the activating Dynabeads®, the cells were able to effectively proliferate, with, in each case, the CAR‐T cells retaining the expression of the CAR gene and their efficacy to target and kill leukaemia cells in vitro. Overall, the two studies 33, 49 showed that fluid dynamic stresses in stirred bioreactors did not damage these cells but that they gave an improved performance compared to static T‐flasks even though λ K at the highest specific power used was of the order of the size of the cell 33.…”

“…Human T‐cells from consenting donors (in one case, after genetic modification to CAR‐T) were grown in static culture (T‐flasks) as a control, in a spinner flask agitated by a magnetic spinner bar and in an unbaffled ambr® 250 bioreactor (Sartorius Stedim Biotech, UK) with a 3‐blade, 45° pitched impeller ( D = 30 mm) at speeds up to 500 rpm. In each case, superparamagnetic Dynabeads®, ∼ 4.5 µm diameter and density ∼ 1.4 kg m −3 , were added to replicate the normal series of events by which T‐cells are activated to protect the body, and without which there would be very poor cell growth 33, 49. These beads are too small to be easily seen in any of the three cases.…”

“…Cells are mainly used in bioprocessing to produce other biological entities such as enzymes or plasmids, either extracellular or intracellular as inclusion bodies by aerobic fermentation. In such cases, the specific oxygen demand varies enormously from entities such as E. coli and Pichia pastoris [32] with very high values to animal cells (e.g., CHO) [7] and CAR-T cells [33], which are much lower as are the cell densities currently achievable. These oxygen demands and cell densities lead to a wide range of k L a values being required (~800 h -1 in the former to~20 h -1 or less in the latter) and a similar wide range of specific powers and air flow rates.…”

“…In the second series of experiments using CAR-T cells [33], speeds up to 500 rpm (1164 Á 10 -4 W kg -1 ) were used. It was found that 200 rpm (74 Á 10 -4 W kg -1 ) gave the highest yields (~5 Á 10 6 cells mL -1 , similar to the T-cells [49]) though statistically the same cell densities were obtained for speeds from 200 to 500 rpm, all higher than the static T-flask control (~2.5 Á 10 6 cells mL -1 ).…”