TRENDS & DEVELOPMENTS IN BIOPROCESS TECHNOLOGY

Seed Train Process Intensification Strategy
Offers Potential for Rapid, Cost-Effective
Scale-Up of Biosimilars Manufacturing
By Rajib Malla, Dhaval D. Shah, Chinmay Gajendragadkar, Vijayalakshmi Vamanan, Deepak Singh,
Suraj Gupta, Deepak Vengovan, Ravi Trivedi, Henry Weichert, Melisa Carpio, and Krishna Chandranoriginally pioneered in the United Kingdom by the process
technology group at Imperial Chemical Industries (ICI) to
reduce their plant size, capital investments, and overhead
costs while increasing productivity.[1]Today, upstream and downstream bioprocess intensification can be achieved in smaller facilities with fewer scale-up
steps to rapidly produce large numbers of therapeutic doses.
An intensified cell culture process could improve overall
manufacturing yields to enable the use of smaller-volume
bioreactors. For example, by increasing the final cell titer by
1 log, a potential scale-up could be reduced from 20,000 L
to 2000 L. This would significantly impact facility footprints, production timelines, media, buffer, reagent, andplant utility costs, as well as allowing the transition fromlarge stainless steel vessels to smaller, more cost-efficient
single-use (SU) bioreactors.
One area where upstream bioprocessing can be intensified is in the seed train. This article discusses how SU
rocking motion (RM) and stirred tank bioreactor technology
is being integrated and tested at leading biosimilars productmanufacturer, Intas Biopharmaceuticals Ltd., Biopharma
Division (Intas), to develop an intensified cell culture seedtrain protocol.
Traditionally, the seed train for production of a biosimilar at Intas has five transfer stages (Figure 1) beginningwith using shake flasks in the post-thaw expansion (N-4)
and then moving through a 5 L rocking motion bioreactor(N-3) to seed 50 L and 500 L stirred tank bioreactors (N-2
and N-1) and ending in a production culture with a final
volume of 4000 L (N). The perfusion approach at N-1 isbeing adopted by several major biopharma companies,
including Bristol-Myers Squibb[2-4] and Roche[5], where cells
stay in the exponential growth phase throughout the entire
culture. Intas has performed a series of studies with SU
RM perfusion bioreactors (working volume 25 L). The goal
was to achieve at least 50 × 106/mL viable cell concentration(VCC) and > 95 % cell viability for seeding the production-scale main bioreactor (4000 L) at a higher initial VCC.

Abstract
perfusion approach at N-1, where cells
stay in the exponential growth phasethroughout the entire culture duration, is
becoming more common as a strategy forprocess intensification. This is because the higher
cell densities it generates allows manufacturers toskip seed stages and reduce process transfer timethrough multiple bioreactor sizes, thus providing
more cost-effective biologics production in smaller
facilities. However, this N-1 perfusion approachrequires optimization. In this article, we describe
the development and proof-of-concept studies withsingle-use rocking motion perfusion bioreactors
in which we have achieved a ten-fold increase in
viable cell count in N-1 seed stage, compared to the
fed-batch control process, in just 6–8 days. We alsomention in detail how we inoculated a 50 L bioreactor production run using this intensified seed train
and show comparable growth kinetics and yieldwith a control process, also at 50 L scale. Using thisintensification approach in the future will help our
manufacturing facility, the Biopharma Division ofIntas Pharmaceuticals Ltd., reach 4000 L production-scale volumes with fewer process transfer
steps, and without changing the feeding strategy
or production bioreactors of our biologics’ portfolio.

A

Introduction
The current costs and global accessibility of biologics andvaccines requires the biopharmaceutical industry to pursue
rapid, yet economic development strategies. One approachfor shortening manufacturing timelines and reducing thecost of goods is “process intensification.” This strategy was
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Seed Train Process Intensification Strategy Offers Potential for Rapid, Cost-Effective Scale-Up of Biosimilars Manufacturing

FIGURE 1. Traditional fed-batch (top) and perfusion (below) seed train cell culture process workflow.

Materials and Methods

Cell Lineto produce cells with high enough densities and viabilities to directly seed a production bioreactor. Additionally,
the Biostat family of RM bioreactors scale from 10–100 L
working volume, making process transfer simpler.
A Biostat STR 50 L bioreactor with SU Flexsafe STR
bags was used in the pilot scale studies. This bioreactor
comes in scales from 50–2000 L. A 4000 L stainless steel
system served as the production bioreactor. The SU bioreactors uses the same established bioreactor design principles
as the 4000 L stainless steel bioreactor, and all the STR bags
have similar geometries across scales[9], which simplifies
process transfer and scale-up.[10]A CHO-S cell line expressing a therapeutic monoclonal
antibody (mAb), which is used as a commercial biosimilar, was chosen for the study. This cell line, its associated(proprietary) media, and culture parameters were selected
for proof-of-concept experiments because the process has
been well-characterized by Intas. Using this clone and media
in an established 6 –7 day fed-batch culture process that
produces a 50–70 ×10 6/mL VCC served as the benchmark
which Intas scientists aimed for when adding perfusion
culture into the seed train.

Bioreactors

Trial Batches

The Biostat® RM 20/50 (Sartorius) was selected for theseed train studies because it is a fully GMP-compliant,rocking motion system where the surface of the medium iscontinuously renewed. This allows mass transfer between
the headspace and medium for efficient mixing with low
shear. SU Flexsafe ® RM and stirred-tank (STR) bags
(Sartorius) were used throughout the entire study. They
are constructed of pharmaceutical-grade, low-density polyethylene with proven cell growth properties as the contact
layer.[6] The Flexsafe RM perfusion bag features an integrated membrane at the bottom for cell-free removal of
media during the culture process, with minimal cell loss ordamage. Studies have shown that RM perfusion bioreactors
can produce very high cell densities (≥ 40–100 ×10 6/mL) at
scales up to 25 L working volume.[7, 8] This makes it possible
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Five seed train trial batch studies were performed to determine the optimum parameters for process intensification.
CHO-S cells were cultured in shake flasks (125–500 mL
volume) and used to seed growth media (5 L) in 10 L SU
bags on the RM 20/50 bioreactor using a range of conditions (Table 1), media volumes, and inoculation densities(Table 2). Cells were sampled manually for analysis via thebioreactor bag’s Luer connection using a sterile SU syringe.
When the VCC reached >2.0–2.5 ×106/mL, perfusion culture
was started by continuous feeding and harvesting from afeed and harvest vessel (Table 3) until the minimum target
VCC of >50 ×10 6 /mL was reached. VCC, cell viability,
metabolites, glucose, and lactate were measured daily (wherepracticable) and used as performance indicators.
2

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Seed Train Process Intensification Strategy Offers Potential for Rapid, Cost-Effective Scale-Up of Biosimilars Manufacturing

Proof-of-Concept Development

perfusion stage, an additional confirmation run (batch 5)
was carried out in the RM bioreactor for inoculation into
a 50 L STR bioreactor (run 3). The culture performance ofcells in the 50 L STR were compared with cells cultured
in a control-seeded 50 L STR batch (inoculated using cells
from a standard fed-batch culture). This served as a proofof-concept to show how cells cultured using perfusion forprocess intensification performed against the traditional
fed-batch seed train expansion methods.

Cells cultured in the RM bioreactor (N-1) from batches 3
and 4 were used to seed two 50 L pilot batches (runs 1 and 2)
in separate SU STR bioreactors. They were operated using
different stir speeds, feed, and media to define the best
culture parameters and yields.

Confirmation Run
After the intensification process was optimized at the N-1

TABLE 1. CHO-S cell culture conditions.
Parameters
Inoculum VCC
Inoculum cell viability
pH post-inoculation
pH setpoint
Temperature
Initial DO
Initial rocker speed(RPM)
Initial rocker angle
Initial aeration flow rate
O 2 (LPM)
Final rocker speed (RPM)
Final rocker angle
Final aeration flow rate
O 2 (LPM)

Batch 1

Batch 2Batch 3Batch 4

Batch 5

0.5 × 106/mL
99%
7.29
7.00
37°C
40%

0.5 × 106/mL
99%
7.14
7.00
37°C
40%

0.5 × 106/mL
99%
7.22
7.00
37°C
40%

0.5 × 106/mL
99%
7.25
7.00
37°C
40 %

0.5 × 106/mL
99 %
7.20
7.00
37°C
40%

16

16

16

16

16

6°

6°

6°

6°

6°

0.1

0.1

0.1

0.1

0.1

22
10°

22
9°

23
9°

23
10°

23
10°

0.2

0.3

0.4

0.7

0.5

NOTE: Dissolved oxygen (DO), revolutions per minute (RPM), and liters per minute (LPM)

TABLE 2. CHO-S cell culture media volumes and inoculation densities.
Parameters
Initial media volume
Inoculum volume
Bioreactor volumepost-inoculation
VCC post-inoculation (at 0 h)
Cell viability post-inoculation(0 h)Batch duration (days)
VCC at harvest
Cell viability at harvest
Target VCC
VCC when perfusion started
Cell viability when perfusion
startedBatch 1
3.7 L
216 mL

Batch 2
3.5 L
200 mL

Batch 33.62 L
271 mL

Batch 4
3.7 L
300 mL

Batch 5
3.6 L
350 mL

4.0 L

3.7 L

3.9 L

4.0 L

4.0 L

0.59 × 106/mL

0.45 × 106/mL

0.45 × 106/mL

0.49 × 106/mL

0.48 × 106/mL

99.0 %

99.5 %

99.5 %

99.5 %

99.2 %

6

10

9

8

6

14.5 × 106/mL
98.0 %
50 × 106/mL

50 × 106/mL
98.9 %
50 × 106/mL

88.5 × 106/mL
97.8 %
50 × 106/mL

150 × 106/mL
97.2 %
50 × 106/mL

64.4 × 106/mL
98.9 %
60 × 106/mL

6.9 × 106/mL

2.6 × 106/mL

2.8 × 106/mL

3.11 × 106/mL

2.3 × 106/mL

99.0 %

98.8 %

99.8 %

99.8 %

98.9 %TABLE 3. CHO-S cell culture feed tank and harvest tank volumes.
Parameters
Total volume of feed
Total volume of harvest

Batch 112.0 L
4.0 L

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3

Batch 222.0 L
3.7 L

Batch 338.4 L
3.64 L

Batch 450.0 L
3.6 L

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Batch 550.0 L
3.6 L

Seed Train Process Intensification Strategy Offers Potential for Rapid, Cost-Effective Scale-Up of Biosimilars Manufacturing

Results
DEVELOPMENT STUDIES

Performance of Batch 1

VCC increased by 2 logs, from 0.45–50.00 ×10 6/mL, and cell
viability was 98.9% at harvest (Table 5). Glucose (measured
from day 3 forward) was maintained at 0.5–2.0 g/L, andlactate decreased from 1.3–0.4 g/L.

During the initial 3 days of this culture run, the VCC andcell viability of the CHO-S cells decreased by 1 log (from0.50–0.06 ×106/mL) and the run was halted. The culture wasremoved, and the bag washed with sterile water. New media(3.5 L) was added (pH 7.05) and this run was restarted. It
was hypothesized that poor culture performance was causedby the cell culture media being sensitive to pH (7.5–7.7),
which led to precipitation and arrested cell growth. For the
rest of this batch run, pH was maintained at 7.0 to determine
if this would improve VCC and cell viability.
During the remaining part of this 6-day culture run,the VCC increased by >1 log, from 0.6–14.5 ×10 6/mL, andmaintained 98% cell viability (Table 4), while glucose andlactate (measured from day 3 forward) were maintained
between 2.0–2.5 g/L.

Performance of Batch 3
To determine reproducibility, cell culture batch 3 was run
for 9 days using similar parameters to batch 2. Perfusion was
not carried out on day 9 (the final day of the run), so no volume
was exchanged. The VCC and CHO-S cell viability increasedby 2 logs, from 0.45–50.00 ×10 6/mL (Table 6) with cells at
98.9 % viability at harvest. Glucose (measured from day 3)
was maintained at 0.50–2.77 g/L, and lactate decreased from
1.3–0.64 g/L. On day 7, 500 mL of batch 3 (perfusion culture)
was taken from the 5.25 L harvested volume to inoculate 38 L
of proprietary media in a 50 L STR bioreactor. Production
run1 reached a peak VCC of 9.76 ×106/mL and 99.2% viability
on day 11. The batch was harvested on day 16 with a VCC of
8.60 ×10 6/mL and 96.6% viability (Table 7).

Performance of Batch 2

During the 10-day culture run of batch 2, the CHO-S

TABLE 4. CHO-S cell perfusion batch 1: VCC and cell viability data from a 6-day run.
Day

InitialMedia Volume

Cell
Viability

Volume Taken/
Harvested

Volume
Added

Perfusion
Rate*

RockerSpeed

RockerAngle

4.00 L

ViableCell Count
0.60 × 106/mL

099.5 %

0L

0L

0

16 RPM

6°

1

3.98 L1.10 × 10 /mL

99.0 %

0L

0L

0

16 RPM

6°

2

3.97 L2.60 × 106/mL

99.0 %

0L

0L

0

17 RPM

6°

3

3.96 L

6.90 × 10 /mL

99.0 %

4L

4L

1

18 RPM7°44.00 L11.00 × 106/mL

99.0 %

4L

4L

1

19 RPM7°54.00 L

15.00 × 10 /mL

99.0 %

4L

4L

1

20 RPM8°64.00 L

14.50 × 106/mL

98.0 %

0L

0L

0

21 RPM

10 °

6

6

6

*Bioreactor volumes per day

TABLE 5. CHO-S cell perfusion batch 2: VCC and cell viability data from a 10-day run.
Day

InitialMedia Volume0

4.00 L

ViableCell Count
0.45 × 106/mL

1

3.98 L0.70 × 106/mL

2

3.97 L

1.50 × 106/mL

3

3.96 L

3.20 × 10 /mL

44.00 L

6.00 × 106/mL

54.00 L

9.91 × 10 /mL

98.8 %

4.00 L6

4.00 L

17.10 × 106/mL

99.3 %

6.00 L7

4.00 L

27.10 × 10 /mL

99.7 %

6.00 L8

3.50 L

40.90 × 106/mL

99.8 %

5.25 L9

3.50 L45.00 × 10 /mL

99.2 %

10

3.50 L50.10 × 106/mL

98.9 %

6

6

6

6

Cell
Viability

Volume Taken/
Harvested

Volume
Added

Perfusion
Rate*

RockerSpeed

RockerAngle

97.0 %

0L

0L

0.0

17 RPM6°

99.0 %

0L

0L

0.0

17 RPM6°

98.5 %

0L

0L

0.0

17 RPM6°

98.8 %

2.00 L2.00 L

0.5

18 RPM7°

99.1 %

4.00 L

4.00 L

1.0

19 RPM7°

4.00 L

1.0

20 RPM8°

6.00 L

1.5

21 RPM8°

6.00 L

1.5

22 RPM9°

5.25 L

1.5

23 RPM9°7.00 L7.00 L

2.0

23 RPM

9°

0L

0L

0.0

23 RPM

9°*Bioreactor volumes per day

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Seed Train Process Intensification Strategy Offers Potential for Rapid, Cost-Effective Scale-Up of Biosimilars Manufacturing
TABLE 6. CHO-S cell perfusion batch 3: VCC and cell viability data from a 9-day run.
Day

InitialMedia Volume03.90 L1

3.88 L2

3.86 L3

3.84 L

ViableCell Count
0.56 × 106/mL

Cell
Viability

Volume Taken/
Harvested

Volume
Added

Perfusion
Rate*

RockerSpeed

RockerAngle

98.7 %

0.00 L0.00 L0.0

16 RPM

6°

ND
2.32 × 106/mL

ND

0.00 L0.00 L0.0

16 RPM6°

4.28 × 106/mL
6.42 × 106/mL

99.7 %

0.00 L0.00 L0.0

16 RPM6°

99.8 %

2.20 L2.20 L

0.5

18 RPM

6°

4

3.82 L5

3.80 L6

3.78 L

12.50 × 106/mL
25.30 × 106/mL

7

3.76 L

41.80 × 10 /mL

99.7 %

8

3.16 L

67.50 × 106/mL

98.7 %

9

3.14 L53.50 × 106/mL

0.00 L6

99.5 %

3.80 L

3.80 L

1.0

19 RPM7°

99.2 %

3.80 L

3.80 L

1.0

19 RPM7°

99.6 %

5.67 L

5.67 L

1.5

20 RPM8°

7.50 L

7.50 L

2.0

21 RPM8°7.00 L7.00 L

2.0

23 RPM9°0.00 L0.0

23 RPM9°92.2 %
NOTE: No data (ND)

*Bioreactor volumes per day

TABLE 7. 16-day production run 1 data: CHO-S cells cultured in the
STR 50 L bioreactor seeded with batch 3 intensified (50 L perfusion) material.
Day

Viable Cell Count

Cell Viability

LactateResidual Glucose

00.36 × 10 /mL
0.64 × 106/mL

99.4 %

0.00 g/L

5.30 g/L

ND

99.5 %

0.25 g/L

5.10 g/L

18.7 mmHg

94.4 %

0.45 g/L

4.87 g/L

24.5 mmHg

30.98 × 106/mL
2.08 × 106/mL

96.3 %

0.77 g/L

4.34 g/L

33.3 mmHg

43.60 × 10 /mL

98.8 %

1.27 g/L

3.70 g/L

32.9 mmHg

599.8 %

1.05 g/L

2.81 g/L

57.2 mmHg

64.85 × 106/mL
6.20 × 106/mL

99.7 %

1.12 g/L

2.14 g/L

31.6 mmHg

77.48 × 106/mL

99.7 %

1.02 g/L

1.54 g/L

37.4 mmHg

88.59 × 106/mL

99.5 %

0.75 g/L

1.26 g/L

43.2 mmHg

99.32 × 106/mL
9.30 × 106/mL

99.6 %

0.56 g/L

1.19 g/L

47.9 mmHg

1
2

10
11
12
13
14
15
16

6

6

pCO2

99.2 %

0.45 g/L

1.02 g/L

44.2 mmHg

9.76 × 106/mL
9.22 × 106/mL

99.2 %

0.23 g/L

1.10 g/L

48.2 mmHg

99.1 %

0.22 g/L

1.02 g/L

51.2 mmHg

9.10 × 106/mL
8.80 × 106/mL

98.8 %

0.25 g/L

1.08 g/L

45.4 mmHg

98.8 %

0.34 g/L

1.01 g/L

39.7 mmHg

8.90 × 106/mL
8.60 × 106/mL

98.8 %

0.41 g/L

1.18 g/L

38.4 mmHg

96.6 %

0.56 g/L

1.16 g/L

37.4 mmHg

mAb Titer Achieved

1.18 g/L

Performance of Batch 4

Development Study Summary

CHO-S cell culture batch 4 was run for a total of 8 days.
All conditions were similar to batches 2 and 3, with the
exception of the perfusion rates, which were increased from
2–3 bioreactor volumes per day. The cells maintained good
viability at 99.2–99.8 % and VCC increased by 3 logs, from
0.49–150 ×106/mL (Table 8), which was more than double thatof batches 2 and 3 at harvest. On day 7, 500 mL of batch 4
material was taken from the 7 L harvested volume to inoculate
38 L of proprietary media in a 50 L STR bioreactor. Production
run 2 VCC reached a peak of 7.70 ×10 6 /mL with 99.20%
viability on day 9. The batch was harvested on day 16 with a
VCC of 6.29 ×106/mL and 91.40% viability (Table 9).

Results from development batches 1–4 showed that the
optimum intensified conditions for culturing CHO-S cells
expressing a mAb biosimilar were: (1) 1–3 volumes per
day of proprietary media in a culture run of 6 –7 days;(2) a rocker speed increase from 16–23 RPM; (3) a rocker
angle of 6–10°; and (4) a pH of 6.9 to prevent loss ofcell viability. These optimized strategies were used forbatches 2 and 3, achieving a VCC of 50–67 ×10 6/mL with
98 % viability. Batch 4, where the perfusion rate wasincreased to 3 bioreactor volumes on day 6, achieved thebest results, with a VCC of 150 ×10 6/mL and viability of
99.3 % (Figure 2).

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TABLE 8. CHO-S cell perfusion batch 4: VCC and cell viability data from an 8-day run.

6

InitialMedia Volume
4.00 L3.98 L
3.97 L
3.96 L
4.00 L
4.00 L
4.00 L7

4.00 L

ViableCell Count
0.49 × 106/mL
0.71 × 106/mL
1.55 × 106/mL
3.11 × 106/mL
6.32 × 106/mL
13.50 × 106/mL
30.67 × 106/mL
68.25 × 106/mL

83.50 L

150.00 × 106/mL

Day
0
1
2
3
4
5

Cell
Viability
99.5 %
99.2 %
99.3 %
99.8 %
99.5 %
99.4 %
99.5 %

Volume Taken/
Harvested
0.0 L
0.0 L
4.0 L
6.0 L
8.0 L
10.0 L
12.0 L

Volume
Added
0.0 L
0.0 L
4.0 L
6.0 L
8.0 L
10.0 L
12.0 L

Perfusion
Rate*
ND
ND
1.0
1.5
2.0
2.5
3.0

RockerSpeed
16 RPM
16 RPM
16 RPM
16 RPM
19 RPM
20 RPM
21 RPM

Rocker
Angle
6°
6°
6°
6°
8°
9°
10 °

99.5 %

12.0 L

12.0 L

3.0

22 RPM10 °99.3 %

11.5 L

11.5 L

3.0

23 RPM

10 °*Bioreactor volumes per day

TABLE 9. 16-day production run 2 data: CHO-S cells cultured in STR 50 L bioreactor seeded with batch 4 intensified material.
Day

Viable Cell Count

Cell Viability

LactateResidual Glucose

00.52 × 10 /mL
0.72 × 106/mL

98.1 %

0.05 g/L

5.36 g/L

19.6 mmHg

99.1 %

0.19 g/L

5.31 g/L

42.2 mmHg

0.91 × 106/mL
2.17 × 106/mL

98.9 %

0.50 g/L

4.55 g/L

ND

1
2
3
4
5
6
7
8
9
10

6

4.20 × 106/mL
5.33 × 106/mL

pCO2

98.2 %

1.10 g/L

3.36 g/L

31.0 mmHg

99.8 %

1.47 g/L

2.65 g/L

22.2 mmHg

98.9 %

34.8 mmHg

1.29 g/L

2.05 g/L

6.75 × 106/mL
7.55 × 106/mL

99.1 %

1.10 g/L

1.39 g/L

22.1 mmHg

98.7 %

0.72 g/L

1.15 g/L

42.5 mmHg

7.12 × 106/mL
7.70 × 106/mL

98.8 %

0.50 g/L

1.03 g/L

37.8 mmHg

98.5 %

0.34 g/L

1.01 g/L

51.9 mmHg

7.35 × 10 /mL
7.21 × 106/mL

98.0 %

0.21 g/L

0.88 g/L

39.5 mmHg

99.2 %

0.23 g/L

1.10 g/L

48.2 mmHg

7.20 × 106/mL
7.02 × 106/mL

97.0 %

0.20 g/L

0.95 g/L

62.7 mmHg

97.0 %

0.08 g/L

0.94 g/L

67.3 mmHg

94.9 %

0.12 g/L

0.88 g/L

29.8 mmHg

156.85 × 106/mL
6.54 × 106/mL

93.6 %

0.34 g/L

1.29 g/L

31.3 mmHg

16

6.29 × 106/mL

91.4 %

ND

ND

ND

12
13
14

Viable Cell Count (106/mL)

11

6

mAb Titer Achieved

1.20 g/L

Batch 1
Batch 2Batch 3Batch 4
Batch 5

Culture Days
FIGURE 2. VCC of CHO-S cells in five perfusion culture development batch runs.
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Seed Train Process Intensification Strategy Offers Potential for Rapid, Cost-Effective Scale-Up of Biosimilars Manufacturing

CONFIRMATION STUDY
Performance of Batch 5

cells expanded via fed-batch (Table 11 and Figures 4A
and 4B). A sparge ventilation was used to remove headspace
CO2 in both runs, but pCO2 levels in the control run oscillated more than the intensified run (Figure 5), as different
proprietary flow rates were used to remove CO2 in each run.
Overall titers (Figure 6) and CQAs (data not shown) of themAb product were also similar, with total galactosylation of
57 % in the mAb derived from the control culture, compared
to 54.5 % from the intensified seed train culture.

Perfusion CHO-S cell culture batch 5 was run for 6 days
with conditions similar to batch 4, which achieved thehighest VCC, as compared to the previous four batches.
The VCC for batch 5 reached 61.2 ×10 6 /mL while maintaining good viability, which was 99.3 % on the final day 6(Table 10). At that point, 500 mL of batch 5 was taken fromthe 9.5 L harvested volume to inoculate 38 L of proprietary
media in a 50 L STR bioreactor. Production run 3 achieveda peak VCC of 9.30 ×10 6/mL with a viability of 95.70% on
day 13. The batch was harvested on day 16 with a VCC of
7.85 ×106/mL and 97.20% viability (Table 11 and Figures 3A
and 3B). These CHO-S cell culture results are comparable inperformance (VCC, viability, glucose, and lactate) to batches
produced by Intas following inoculation with the control

Confirmation Study Summary
CHO-S cells cultured in the SU 50 L STR bioreactor
performed well, following inoculation with the intensified(perfusion) seed train product, indicating that this approach
can be used for a hybrid perfusion/fed-batch process scale-up.

TABLE 10. CHO-S cell perfusion batch 5: VCC and cell viability data from a 6-day run.
Day
0
1
2
3
4
5
6

InitialMedia Volume
4L
4L
4L
4L
4L
4L
4L

ViableCell Count
0.48 × 106/mL
1.15 × 106/mL
2.30 × 106/mL
5.35 × 106/mL
12.40 × 106/mL
26.50 × 106/mL
61.20 × 106/mL

Cell
Viability
99.0 %
98.5 %
98.8 %
99.4 %
99.3 %
99.4 %
99.3 %

Volume Taken/
Harvested
0.0 L
0.0 L
0.0 L
3.9 L
5.9 L
7.6 L
9.5 L

Volume
Added
0.0 L
0.0 L
0.0 L
3.9 L
5.9 L
7.6 L
9.5 L

Perfusion
Rate*
ND
ND
1.0
1.5
2.0
2.5
ND

RockerSpeed
16 RPM
16 RPM
17 RPM
18 RPM
19 RPM
20 RPM
20 RPM

Rocker
Angle
6°
6°
7°
8°
9°
10°
10 °

*Bioreactor volumes per day

TABLE 11. 16-day production run 3 data: A comparison of CHO-S cell culture performance using STR 50 L bioreactors seeded
separately with the control (used as seed to inoculate the 4000 L fed-batch process) and intensified (50 L perfusion) materials.
Day

VCC(× 10 6/mL)

Cell Viability(%)

Accumulated
Lactate (g/L)

Residual
Glucose (g/L)

pCO2(mmHg)

Control

50 L

Control

50 L

Control

50 L

Control

0.52
0.72
0.91
2.17
4.20
5.33
6.75
7.55

0.60
1.20
2.81
5.45
6.88
7.52
7.55
8.02

98.11
99.10
98.91
98.19
99.76
98.89
99.12
98.69

50 L
99.90
99.40
99.60
99.20
99.80
99.70
99.60
99.60

Control

0
1
2
3
4
5
6
7

0.05
0.19
0.50
1.10
1.47
1.29
1.10
0.72

0.04
0.19
0.77
1.15
1.17
0.96
0.74
0.50

5.36
5.31
4.55
3.36
2.65
2.05
1.39
1.15

5.29
5.25
4.07
3.02
2.97
2.50
2.43
1.95

19.60
42.20
ND
31.00
22.20
34.80
22.10
42.50

50 L
45.70
87.10
34.40
25.70
32.40
38.50
52.90
45.80

8

7.12

9.12

98.80

99.20

0.50

0.32

1.03

1.66

37.80

52.70

9
10
11
12
13
14
15
16

7.70
7.35
7.21
7.20
7.02
6.85
6.54
6.29

9.06
8.72
9.12
8.51
9.30
8.19
7.91
7.85

98.50
98.00
97.00
97.00
95.70
94.90
93.60
91.40

99.60
99.60
99.20
99.20
99.10
99.00
98.90
97.20

0.34
0.21
0.20
0.08
0.09
0.12
0.34
ND

0.21
0.10
0.07
0.07
0.08
0.09
0.10
0.12

1.01
0.88
0.95
0.94
0.99
0.88
1.29
ND

1.30
1.37
1.01
1.27
1.10
1.20
1.22
1.20

51.90
39.50
62.70
67.30
52.90
29.80
31.30
ND

49.80
54.20
55.50
52.10
49.80
51.20
53.20
50.80

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mAb Titer
Achieved (g/L)
Control

50 L

1.34

1.42

Seed Train Process Intensification Strategy Offers Potential for Rapid, Cost-Effective Scale-Up of Biosimilars Manufacturing

Viable Cell Count (106/mL)

A

Culture Days
Control batch

50 L batch (intensified seed batch)

Cell Viability (%)

B

Culture Days
Control batch

50 L batch (intensified seed batch)

FIGURE 3. 16-day production run 3 data comparing: (A) VCC; and (B) cell viability in CHO-S cells
cultured using STR 50 L bioreactors seeded separately with the control and intensified materials.

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Seed Train Process Intensification Strategy Offers Potential for Rapid, Cost-Effective Scale-Up of Biosimilars Manufacturing

Residual Glucose (g/L)

A

Culture Days
Control batch

50 L batch (intensified seed batch)B

1.6

Lactate Accumulation (g/L)

1.4
1.2
1.0
0.8
0.6
0.4
0.2
0

Culture Days
Control batch

50 L batch (intensified seed batch)

FIGURE 4. 16-day production run 3 data comparing: (A) residual glucose; and (B) lactate accumulation in
CHO-S cells cultured using STR 50 L bioreactors seeded separately with the control and intensified materials.

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pCO2 (mmHg)

Seed Train Process Intensification Strategy Offers Potential for Rapid, Cost-Effective Scale-Up of Biosimilars Manufacturing

Culture Days
Control batch

50 L batch (intensified seed batch)

FIGURE 5. 16-day production run 3 data comparing pCO2 in CHO-S
cells cultured using STR 50 L bioreactors seeded separately with thecontrol and intensified materials.

Control batch

50 L batch(intensified seed batch)

FIGURE 6. 16-day production run 3 data comparing
CHO-S cell mAb titers using STR 50 L bioreactors seeded
separately with the control and intensified materials.

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Seed Train Process Intensification Strategy Offers Potential for Rapid, Cost-Effective Scale-Up of Biosimilars Manufacturing

Discussion
At Intas, when using a standard fed-batch seed train perfusion culture intensification.[7, 8]
production protocol, the VCC typically reaches 7–8 ×106/mL
These results indicate that high cell densities can beafter 14 –16 days of fed-batch culture with the CHO-S clone achieved in less time using perfusion culture in the seed
and media used as a control in this study. By intensifying train. Production runs incorporating intensified seed
the seed train cell expansion via perfusion technology, it is trains also have comparable growth kinetics and yields, aspossible to achieve a ten-fold increase in VCC at N-1 seed compared to the control seed train process at 50 L scale.
stage compared to the control batch just described, in only By using an RM 50 L perfusion bioreactor to generate cell
6–8 days. This is consistent with other studies where very batches at a VCC of 50 ×10 6/mL, scientists at Intas could
high cell densities (≥ 40 –100 ×10 6 /mL) scalable up to a potentially seed a 4000 L bioreactor with 25 L of this mateworking volume of 25 L are possible with CHO cells using rial to achieve a target inoculation VCC of 0.4 ×10 6/mL.
Conclusion
The development and proof-of-concept studies detailed productivity while consuming fewer resources. Other posiin this article have shown that using SU RM perfusion tives include reducing the manufacturing footprint withoutbioreactors to produce seed train material could potentially changing the production bioreactor or feeding strategy,
help Intas achieve process intensification up to 4000 L with potentially delivering the products in Intas’ biologics portfewer process transfer steps. This could measurably increase folio more cost-effectively.
References[1] Reay D, Ramshaw C, Harvey A. A brief history of process intensification. In: Process intensification, 2nd edition. Butterworth-Heinemann,
2013, pp 1–20. https://www.elsevier.com/books/process-intensification/
reay/978-0-08-098304-2[2] Yongky A, Xu J, Tian J, Oliveira C, Zhao J, McFarland K. Processintensification in fed-batch production bioreactors using non-perfusion
seed cultures. mAbs, 2019; 11(8): 1502–14. https://doi.org/10.1080/1942
0862.2019.1652075[3] Xu J, Xu X, Huang C, Angelo J, Oliveira CL, Xu M. Biomanufacturing
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10.1080/19420862.2020.1770669[4] Morrow KJ. Continual improvements advance continuous biomanufacturing. GEN, 2019. https://www.genengnews.com/topics/bioprocessing/continual-improvements-advance-continuous-biomanufacturing/[5] Pohlscheidt M, Jacobs M, Wolf S, Thiele J, Jockwer A, Gabelsberger J,
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Int, 2014. https://bioprocessintl.com/sponsored-content/new-f lexsafe-bag-family-combining-material-science-film-expertise-qbd-reliable-consistent-cell-growth/[7] Yang WC, Lu J, Kwiatkowski C, Yuan H, Kshirsager R, Ryll T,
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About the Authors
Rajib Malla , Dhaval D. Shah , Chinmay Gajendragadkar1, Vijayalakshmi Vamanan1, Deepak Singh, PhD1,
Suraj Gupta2, Deepak Vengovan2, Ravi Trivedi2, Henry Weichert3, Melisa Carpio4, and Krishna Chandran5
1. Intas Pharmaceuticals Ltd., Biopharma Division, Ahmedabad, Gujarat, India
2. Sartorius, Ahmedabad, Gujarat, India
3. Sartorius, Göttingen, Germany
4. Sartorius, San Francisco, California USA
5. Sartorius, Bangalore Urban, Karnataka, India
1

1

Corresponding Authors:
Deepak Vengovan (Deepak.Vengovan@Sartorius.com), Manager of Field Application Specialist CCT.
Suraj Gupta (Surajkumar.Gupta@Sartorius.com), Field Application Specialist CCT.
Tel: +91.79.6616.8082
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