SUPPLIER SIDE

Intensified Seed Train Strategy forFaster, Cost-Effective Scale-Up ofBiologics Manufacturing
Jens-Christoph Matuszczyk, Johannes Lemke, Markus Schulze,
Sabrina Janoschek, Gerben Zijlstra, and Gerhard Greller

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RM2 VCD

RM10 VCD

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RM50 VCD

RM2 viability

RM10 viability

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drive seed ratios of >1:100. That allows
skipping of process-transfer steps. For
example, HCD cell banks can bethawed directly and inoculated into RM
bioreactors at higher volumes (0.5–2 L
depending on the HCD cell-bankvolume), thus bypassing cell-culture
expansion in shake flasks and thepotential errors and contamination
issues associated with manual handlingof flasks. Also, with perfusion in theN – 1 seed train, cells can reach higher
densities for inoculating a pilot or
production bioreactor directly without
process transfer through multiple
intermediate-scale STR bioreactors.
That reduces both the number of steps
and the total number of seed-train days,
thus reducing initial investment andoperational CoG. However, not all CHO
clones that produce MAbs can becultured in intensified processing

NOVEMBER–DECEMBER 2020

Viability (%)

62 BioProcess International

Figure 1: Viable cell density (VCD) and viability of Chinese hamster ovary (CHO) cells
cultured in RM perfusion bioreactors with working volumes from 2 L to 50 L

Viable Cell Density (106 cells/mL)The high costs of and limits on
global accessibility of biologics
such as monoclonal antibodies(MAbs) are focusing thebiopharmaceutical industry’s attention
on strategies for rapid, economicaldevelopment of such therapies. Processintensification is one approach to help
shorten manufacturing timelines andreduce cost of goods (CoG) (1, 2). Today,
process intensification in upstream cell
culture enables biologics manufacturing
in facilities with smaller footprints andlower scale-up volumes than waspossible before. Intensified processingof Chinese hamster ovary (CHO) clones
that produce MAbs is being developedin the seed train of upstream cell
cultures (3) to support generation ofhigh–cell-density (HCD) cell banks andprocesses to reduce plant size, capital
investment, and overhead costs while
increasing productivity.
In traditional practice, MAb
manufacturing relies on seed ratios of~1:5 (3) with an initial inoculum of 0.3 ×
106 cells/mL (4) using a seed train of fiveto eight transfer steps. Standard seedtrains typically begin with shake flasks
in the postthaw expansion (N – 8 toN – 5) and then move through arocking-motion (RM) 5–50-L bioreactor(N – 4 to N – 3) to seed a series of N – 2
to N – 1 stirred-tank (STR) bioreactors(200 L and 500 L) ending in a production
culture (N) with a volume of 2,000 L.
Adopting an intensified cell culture
approach at N – 4 to N – 1 by, forexample, using 2D RM bioreactors inperfusion mode (where cells stay in the
exponential growth phase throughout
the entire culture) could increase viable
cell densities to 100 × 106 cells/mL and0because high cell densities cancompromise protein expression levels
and genetic stability (5).
Our goal was to explore thepossibility of culturing a CHO clone that
expresses a commercial MAb
therapeutic in HCD for an intensified,scalable cell-culture seed-train protocol.
We investigated whether single-use RM
perfusion bioreactors (working volume
1–25 L) could achieve a reproducible
cell culture with at least 100 × 106/mL
viable cell density (VCD) at different
working volumes. The HCD culture wasmonitored using process analyticaltechnology (PAT) at all scales. When theculture in the RM bioreactor (1-L workingvolume) reached the required HCD, aculture volume was used to seed apilot-scale STR single-use bioreactor(200 L) automatically, which then ran infed-batch mode. During the pilot run,
SPONSOREDCell Line: We chose a CellcaCHO-DG44
cell line expressing a commerciallyavailable human MAb to investigate thepracticality of using HCD perfusion
culture to inoculate a pilot-scalebioreactor. The cell line, with its
associated media and cell-culture
parameters, was selected for proof-ofconcept studies because the fed-batchprocess used to produce this MAb has
been well-characterized by Sartorius.
Bioreactors: The Biostat RM 20/50
bioreactor (Sartorius) was chosen forseed-train studies because it
continuously renews the cell-culture
medium, enabling mass transfer
between the headspace and medium
for efficient, low-shear mixing. The
system was used with single-use
Flexsafe RM perfusion bags (1–25-Lworking volume) constructed ofpharmaceutical grade low-density
polyethylene, which is used throughout
the entire range of RM and STR
bioreactor bags to ensure consistentquality. The bags each feature an
integrated perfusion membrane at thebottom, forming a compartment forremoving cell-free media during theperfusion process for minimal loss of ordamage to the cells. The RM perfusionbioreactors scale from 2 L to 50 L
(working volume 1 L–25 L) using acommon RM bioreactor design tosimplify process transfer.
A Biostat STR bioreactor (200 L) withsingle-use Flexsafe STR bags was usedas the pilot-scale single-use bioreactor.
The Sartorius STR bioreactor range
scales from 50 L to 2,000 L and uses
the same established bioreactor designprinciples as stainless-steel bioreactors.
All bags have similar geometries across
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Viable Cell Density (106 cells/mL)

Materials and Methods

Figure 2 Comparison of VCD and viability of CHO cells in 200-L pilot-scale fed-batch
culture inoculated with cells from standard or intensified seed trains

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we measured cell density and viability
along with the MAb’s critical quality
attributes (CQAs), then compared thoseresults with the same MAb produced
through a standard seed train. The
results established whether a HCD
perfusion seed train can inoculate astandard manufacturing N-stagebioreactor to reduce the number oftransfer steps and whether it is possible
to use a PAT-based control regime for
automated inoculation of N-stage
processes to reduce inoculation
volumes.

10024VCD init. seed
STR20068Time (days)

Via int. seed
STR200VCD std
STR200scales (5) to facilitate process transfer
and scale-up (4).

Proof-of-Concept Studies

Seed Train Study: To demonstrate theperformance and scalability of cultures
in RM perfusion bioreactor bags, wecultured CHO cells for six days inperfusion bioreactor bags of 2 L, 10 L,20 L, and 50 L (working volumes of 1 L,5 L, 10 L, and 25 L) using proprietarymedia and feeds at a minimum cellspecific perfusion rate (CSPR) of 50 PI(cells/day). The cells were cultured at
36.8 °C, pH 6.95, and DO 60%(maintained by increasing the rocking
rate at a 10° angle). On-line PAT
sensors, including the BioPAT ViaMass
sensor, were used to monitor andcontrol the culture’s VCD and viability
and enable automatic feeding. The 2-L
culture was cultivated for an additional
two days under perfusion conditions todetermine a maximum VCD.
Pilot Bioreactor Studies: A VCD modelfrom the RM perfusion bioreactor wascreated from the signal from the BioPAT
ViaMass sensor. With this model, theVCD was measured on-line constantly,
and when cells reached a trigger pointof 100 × 106 cells/mL, a cell inoculum
volume calculated to produce an initialcell density of 0.33 ± 0.04 × 106 cells/mL
was transferred automatically from theRM perfusion bioreactor to the pilot
scale STR bioreactor. Cells cultured inthe perfusion bioreactor (400 mL at

10120

Via std
STR200approximately 100 × 106 cells/mL) wereused to seed a Biostat STR 200-L
single-use bioreactor. Cells werecultured for 12 days using proprietary
media and feeds at 36.8 °C, pH 7.1, andDO 60%. Feeds were added after athree-day batch phase on day 4. VCD
and cell viability data were measured
and collected by the BioPAT ViaMasssensor. A cell free supernatant was
sampled for N-glycan profiling todetermine MAb product quality andtiter. The results were compared withhistorical data from a standard seedtrain for which cells were cultured instandard Biostat RM 20/50 bags (14 L atapproximately 3 × 106 cells/mL) andused to inoculate a Biostat STR 200 L
single-use bioreactor.

Results and Discussion

Performance of Seed Train: The results(Figure 1) show that in six days, the RM
perfusion bioreactors can grow CHO cell
cultures with >90% viability and with highcell densities of >100 × 106 cells/mL. That
represents a 33-fold increase in VCD
compared with an inoculum from astandard RM bioreactor. Over eight days,in a 2-L bag, these perfusion bioreactors
support cell culture to VCDs of 170 × 106
cells/mL with >90% viability,demonstrating that cellular oxygen
demand can be supported at such highcell densities.
Our VCD and viability results are
consistent at all scales tested up toNOVEMBER–DECEMBER 202018(11–12)

BioProcess International63

5.0

100

4.590

4.080

profiles. Our results indicate that using
this intensified seed-train approach canincrease productivity withoutcompromising product quality andcould potentially increase the costeffectiveness of biologics production.

3.570

References

3.060

2.550

2.0

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STR Fed-Batch Standard SeedG1/G0 ratio

64 BioProcess International0STR Fed-Batch Intensified Seed

G2/G0 ratio

50 L. The reproducibility and scalabilityof HCD cultures using the perfusionbioreactors shows that this method canproduce cells with high enough VCDs
to seed 200-L pilot bioreactors directly,
indicating the potential for inoculating
2,000-L production bioreactors using asuitable HCD culture, e.g., by using a
RM 10 or 20.
Pilot-Scale Study: Cells in proprietary
media from the RM perfusion bioreactor
were used to seed a 200-L pilot-scalebioreactor using an automated PAT
sensor-controlled inoculation set with atarget initial cell density of 0.33 ± 0.04 ×
106 cells/mL. That resulted in an actual
starting cell density of 0.37 × 106 cells/mL, demonstrating that the PAT sensor
can achieve inoculation precisioncomparable with manual seeding. In
total, 0.4 L of culture from theintensified seed train was used toinoculate the pilot-scale bioreactor
compared with needing over 13 L of cell
culture from a standard seed train.
Thus, using HCD cells to seed the pilotscale bioreactor required over 30-foldless inoculation volume than whenusing cells from a standard seed train.
Fed-batch results (Figure 2) fromthe pilot-scale bioreactor seeded withHCD cells from the RM perfusionbioreactor and the traditional fed-batch
cell culture show comparable growth
characteristics over the entire processrun. Both achieve a maximum VCD of
25 × 106 and viability of >90% in sevendays. The MAb titers also were similar,
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Fucosylation (%)

Ratio

Figure 3: Comparison of MAb critical quality attributes (CQAs) expressed by Chinese
hamster ovary (CHO) cells in 200-L pilot-scale fed-batch culture inoculated with cells fromstandard or intensified seed trains

Fucosylated forms (%)

with 3.5 g/L achieved using thestandard seed train compared with
3.2 g/L from the intensified seed train.
Those results indicate that using HCD
cells to seed pilot-scale bioreactors
has no adverse effect on cell growth,viability, or titer over a standard 12-day
fed-batch run at 200-L scale in asingle-use STR bioreactor.
Product Quality: CQAs of the MAb
product from fed-batch cultures
inoculated with standard and fromintensified seed trains (Figure 3)
showed similar trends in glycan profilesof G2/G0 and G1/G0 ratios and with
95% fucosylated forms produced. The
performance results of the cells
cultured in the pilot-scale bioreactor
from the intensified seed train showsthat using HCD cells to seed a pilotscale bioreactor does not affect productquality. That indicates the usefulness of
this approach for a hybrid perfusion/
fed-batch process scale-up.

Productivity and Quality

The proof-of-concept studies presented
here show that single-use RM perfusionbioreactors can achieve HCD cell
cultures (>100 × 106 cells/mL) with >90%
viability at every scale from 1 L to 25 L
working volumes. The studies also
demonstrate that HCD cells (inoculatedby means of automated process control)and cells from a standard fed-batch
culture used to seed a 200-L STR
bioreactor produce comparable cell
growth, viability, titer, and MAb CQANOVEMBER–DECEMBER 2020

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Corresponding author Jens-ChristophMatuszczyk is manager, bioprocessing
upstream, Sartorius, Göttingen, Germany;
49-551-308-4245; Jens.Matuszczyk@sartorius.com. Johannes Lemke is ascientist and Markus Schulze andSabrina Janoschek are PhD students.
Gerben Zijlstra is a global technology
consultant, and Gerhard Greller is asenior scientist at Sartorius Stedim
Biotech GmbH. Editorial contact: Dominic
Grone, Sartorius; 49-551-308-3324;
dominic.grone@sartorius.com.
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