1 Introduction
Lentiviral vectors (LVV’s) are one of the fastest growing vectors under
development within the cell and gene therapy industry (McCarron,
Donnelley, McIntyre, & Parsons, 2016). They offer many advantages
including the ability to transduce dividing and non-dividing cells,
offer high transduction efficiency, and low immunogenicity (Kotterman,
Chalberg, & Schaffer, 2015). These favourable characteristics make them
a promising tool for use in the treatment of various genetic and
acquired diseases such as beta-thalassemia (Cavazzana-Calvo et al.,
2010), Parkinson’s disease (Palfi et al., 2014) as well as oncology
(Levine, 2015). With the increasing use of LVV in translational research
and clinical programs, robust and scalable production processes are of
critical importance in view of implementing these novel therapies for
routine use (Merten, Hebben, & Bovolenta, 2016). However, current LVV
batch or fed-batch upstream processes are limited by the inability to
achieve high cell densities with the HEK293T cell line, resulting in
poor yields (Manceur et al., 2017). Optimisation of cell culture
conditions is therefore necessary to improve upstream efficiency.
Initial optimisation of suspension cell culture is traditionally carried
out at small scale (i.e. microlitre to millilitre scale) under
conditions that aim to mimic the large-scale bioreactor environment
(Kumar, Wittmann, & Heinzle, 2004), and where possible, at
high-throughput. However, adopting such an approach for LVV production
employing conventional scales (e.g. miniature stirred tank reactors
(STRs) and shake flasks) is both costly and impractical. Microwell based
systems offer a suitable alternative to obtain key process design data
early and cost-effectively (Martina Micheletti & Lye, 2006).
Previous studies have demonstrated the use of 24-standard round well
(24-SRW) microtiter plates for the suspension cultivation of mammalian
cell lines such as hybridoma (Barrett, Wu, Zhang, Levy, & Lye, 2010; M.
Micheletti et al., 2006) and Chinese Hamster Ovary (CHO) (Chaturvedi,
Sun, O’Brien, Liu, & Brooks, 2014; Mora et al., 2018; Silk et al.,
2010) for monoclonal antibody production. However, examples of their use
for LVV production are few in literature. Guy et al. , 2013
describe cultivation of HEK293 cells in 24-SRW plates to establish
operating conditions for a 0.5 L wave bioreactor culture and
demonstrated that the 24-SRW is an effective scale-down model (Guy,
McCloskey, Lye, Mitrophanous, & Mukhopadhyay, 2013). HEK293T stable
cell line cultivation in 24-deep square well (24-DSW) plates has yet to
be demonstrated in literature. Compared to the 24-SRW plate, the 24-DSW
format is advantageous as it offers 3-6 times greater working volume.
Studies have also shown that square shaped wells have a better mixing
and oxygen transfer profile compared to round well plates (Wouter A.
Duetz & Witholt, 2004). To adopt the microwell system as a scale-down
tool for LVV production, cell culture performance such as growth
kinetics and LVV productivity must be matched with bench to pilot scale
bioreactor processes. If not, there is no guarantee that the optimised
process conditions achieved using microwells will be maintained upon
scale-up.
Scale translation in cell culture is notoriously challenging due to the
number and complexity of the variables affecting the physical and
biological process. Criteria such as gas-liquid mass transfer
coefficient (kLa), power input per unit volume (P/V) and
mixing time have been used in chemical processes and adopted in
biochemical engineering for bioreactor scale translation for different
cultures (Alvin W. Nienow, 2006). Matched kLa is often
used for scaling cell cultures with high oxygen demand (Ferreira-Torres
et al., 2005). The oxygen demand for mammalian cells (e.g. HEK293) is
relatively low compared to bacterial cell lines (e.g. E. coli) (Alvin W.
Nienow, 2006), therefore matched kLa was not considered
for the HEK293T stable producer cell line cultures. The use of matched
P/V in this study was limited by the lack of accurate methods of
determining P/V in microwells. Current methods only provide estimates of
the power input using computational fluid dynamics (CFD) predications of
the mean energy dissipation (Zhang, Lamping, Pickering, Lye, & Shamlou,
2008). As there are no direct methods to compare against, the CFD
predictions are yet to be validated as accurate models of the actual
P/V. In comparison, there are now more accurate and semi-automated
methods of measuring mixing time such as the dual indicator system for
mixing time (DISMT) technique. This method also enables mixing time to
be characterised across reactors with different scales and geometries.
Furthermore, Sani and Baganz, 2016 demonstrated matched mixing time as a
suitable criterion for scaling between 0.5 L micro-bioreactor (MBR) and
5 L STR for a GS-CHO cell line (Sani & Baganz, 2016). However, scale
translation for smaller scale vessels with different geometries is yet
to be established. Therefore, in this work, we evaluate mixing time as a
basis for HEK293T stable LVV producer cell line culture
scale-translation between 24-DSW microtiter plates and bench-scale 2 L
STR. First, we characterise mixing times in the 2 L STR and 24-DSW
plates using the DISMT technique. Thereafter, growth kinetics, LVV
productivity and cell metabolism of the HEK293T stable producer cell
line are compared between the microwell and STR cultures at matched
mixing time.