A Strategic Approach To Selecting the
Optimal Process Intensification Scenario

A Strategic Approach
To Selecting the Optimal Process
Intensification Scenario
Priyanka Gupta and Katy McLaughlinCurrent demands placed on thebiopharmaceutical industry are pushing
manufacturers toward intensified processing.
Process intensification (PI) describes anoverall approach that modifies unit operations or an
entire manufacturing process to optimize efficiency.
Biomanufacturers can measure improvements by anumber of approaches, including reducing operation
costs, speeding up production, increasing yields, andenhancing flexibility (1).
Intensification strategies can be applied toindividual unit operations or throughout an entire
facility or process, using an end-to-end approach. The
three intensification scenarios typically used in
upstream processing are seed-train intensification(usually at the N – 1 stage), concentrated fed-batch
production, and dynamic perfusion (at the productionbioreactor stage) (Figure 1 and Table 1). In downstream
processes, intensification strategies typically involve
moving from single-column to multicolumn
chromatography (MCC).
Here, we discuss how adopting single-use
intensified manufacturing satisfies key businessdrivers. We then provide a process-decision matrix tohelp simplify the implementation of PI in new andexisting facilities. Finally, we present an example
illustrating how upstream PI can increase productivity
and reduce costs without compromising quality (2).

Process Intensification: A Decision Matrix

Benefits of Intensified Production: The philosophy of PI
creates improvements that help manufacturers keep up
with growing demands, allowing them to stay ahead of
their global competitors.
First, by lowering the need for high upfront
investment, PI helps mitigate the risk of failure during
early biopharmaceutical development when it is notalways clear which molecules will make it to market.
Therefore, when building a facility to support
production of a new biologic, it is important to consider
options that require low upfront investment andlimited expenditure on large equipment (such as
multiple commercial-scale bioreactors). With PI, you
2

BioProcess International

19(10)si

October 2021

can limit the amount of equipment needed to build aproductive facility (Table 1).
In the highly competitive biopharmaceutical space,
the first product to market has a significant advantage.
PI can reduce the time it takes to get into clinicalstudies and/or commercial sales by accelerating
development. The build-out time for a good
manufacturing practice (GMP)–compliant facility is
typically around four years, reduced to two years orless when PI strategies are implemented (Table 1).
Intensification also can reduce a facility footprint by
50–70%, creating a smaller, more efficient facility(Table 1). That can help developers save on overhead
costs such as energy use and facility maintenance. A
reduced footprint helps companies achievesustainability targets, such as by lowering the processmass intensity index (PMI), electricity use, and water
consumption. Such changes reduce the overall carbon
footprint, an increasingly important factor in thecurrent environment.
Finally, PI can increase productivity by two- tothreefold (Table 1). The enhanced efficiency reduces thecost of goods by up to 30% or more, depending on the
combination of approaches that are implemented
throughout the process.
Process Decision Matrix: Once a drug developer has
decided that intensification can benefit its facility, it
must select the scenario(s) most compatible with its
processes. Decision-making will be shaped by manyfactors, including the drug candidate, market, facility,
and operations; there is no universal solution. Below isa simplified framework for selecting an optimal
strategy for your facility.
Facility Throughput — 300 kg/Year or Less: Usually, thefirst driver to begin the decision-making process will befacility throughput (Figure 1). For facilities where
throughput is under 300 kg/year, seed-train
intensification or dynamic perfusion will typically be
the optimal PI approaches.
An N – 1 perfusion, high-inoculum fed batch canincrease productivity by up to twofold and can be
implemented easily into an existing process, making it
an excellent transition into the philosophies of PI.
Sponsored

Table 1: Comparing different upstream process-intensification (PI) strategies with standard fed-batch production, in
which harvest happens only at the end of production; N – 1 perfusion (seed-train intensification) reduces the timeneeded to reach the cell densities required to populate a production bioreactor. Concentrated fed-batch production
involves continuous circulation of media, but the product is harvested only at the end of a procedure. Finally,
dynamic perfusion involves continuous feed and harvest of the product through microfiltration.
Benefits/Disadvantages
Compared with Fed Batch

N – 1 Perfusion
High-Inoculum Fed-Batch

Concentrated Fed-Batch

Dynamic Perfusion

—

Yes

Yes

Productivity increase*

Up to ×2

Up to ×4

Up to ×5 in 12–20 days

Culture time saving

Up to 5 days in seed train

Up to 10 days in seed train **Up to 10 days in seed train **Investment CoGS

Lower

Lower

Lower

Similar throughputs with single
use and with 10-kL stainless steel

Media use

Slightly higher

High

High

Alternatingtangential flow (ATF)

Yes, microfiltration

Yes, ultrafiltration

Yes, microfiltration

Batch scheduling

Short batches, flexible USP to
DSP scheduling (multiproduct)

Short batches, flexible USP to
DSP scheduling (multiproduct)

Less flexible, longer batches,
DSP 1:1 connected toperfusion permeate*typical numbers**with 5-mL HCD vials and RM perfusion for N – 2 and N – 1

Additionally, the increase in media consumption ismodest (Table 1) (3).
Dynamic perfusion, followed by MCC in thedownstream process, has the potential to increase
productivity by up to five-fold in under three weeks(Table 1), and can significantly reduce facility footprint.
However, dynamic perfusion will increase media use
by over 1,000%, which will be an importantconsideration in facility design (3).
Facility Throughput — 500 kg/Year or More: Where
throughput is over 500 kg per year, manufacturers will
benefit most from concentrated fed-batch or N – 1
perfusion. Concentrated fed-batch is highly productive(up to 4× increase) and flexible, making it suitable formultiproduct facilities. However, it requires
significantly more culture media than standard fedbatch production does (Table 1) (3). As discussed for
low-throughput processes, N – 1 perfusion also
increases productivity (about 2×) with limited process
disruptions and upfront investment. Beyond increasing
overall productivity, applying PI strategies to highthroughput processes can reduce facility footprint by
50% or more, significantly improving efficiency.
Facility Requirements: After considering facility
throughput, manufacturers will need to consider how
PI fits in with their facilities and operations.
Intensification in Existing Facilities: Approaches toimplementing PI will differ between new and existing
facilities. Facilities that are not yet in operation havemore options because designers do not need to consider
how changes affect ongoing production.
Seed-train intensification is an effective strategy
that manufacturers use to prevent the need forSponsoredsignificant changes to their existing operations andfacility set ups. Although N – 1 perfusion increases
upstream productivity, it does not affect product titers
enough to require significant changes to downstream
procedures. Seed-train intensification also will notchange media and buffer requirements dramatically,
does not involve additional expertise, and can be
implemented seamlessly into an existing facility.
Converting to a Single-Use Facility: As current trends
push manufacturers toward intensified bioprocesses,
the same pressures drive facilities toward single-use(SU) manufacturing. Comprehensive SU platforms
promote consistency and robustness in the process
while providing the flexibility needed to respond tochanges quickly. A company using traditional stainlesssteel equipment might want to combine thephilosophies of PI with SU technologies to create a nextgeneration facility.
Concentrated fed-batch production could be an
excellent strategy to move away from stainless-steel
facilities toward SU platforms. This strategy is mostsuitable for high-throughput production and offers
flexibility for multiproduct manufacturing.
Sensitive Modalities: Many emerging modalities are
sensitive biological molecules, such as bispecificmonoclonal antibodies (bsAbs), which require
adjustments to the production process for traditional
biotherapies. For example, some modalities are notcompatible with a standard fed-batch process in
production.
Dynamic perfusion is a superior choice for unstableor difficult-to-express molecules. Also compatible withrelatively small facilities implementing a modular
October 2021

19(10)si

BioProcess International3

Figure 1: Process-decision criteria for PI implementation are included in a flow chart to simplify the selection of an
intensification strategy. Upstream approaches include N − 1 perfusion high inoculum fed-batch, concentrated fedbatch, and dynamic perfusion. Downstream approaches include multicolumn chromatography (MCC).
Throughput

Standard Process

25−300 kg/year

500 kg/year or higher

Fed-Batch

Fed-BatchConcentrated Fed-Batch —Batch/MCC Capture

Highest Productivity

Lowest CoGSN − 1 Perfusion High
Inoculum, Fed-Batch —Batch/MCC Capture

Flexible/Smaller
Footprint, Lower
Upfront Investment

Dynamic Perfusion
MCC Capture

approach to PI, dynamic perfusion offers flexibility for
manufacturing multiple molecule types.

Intensified Seed-Train Strategy for Effective
Scale-Up of Biologics Manufacturing

Seed Intensification — Proof of Concept: Seed-train
intensification is an ideal first step in exploring thebenefits of PI. Focusing on the seed train offers asignificant increase in productivity without making
substantial changes to the existing facility set-up.
The following article, first published in BioProcess
International in December 2020, is a proof-of-concept
study demonstrating the feasibility and value of seedtrain intensification. Using Chinese hamster ovary(CHO) cell cultures grown in single-use bioreactors,
our researchers investigate the feasibility of
populating a production bioreactor with high-density
cell cultures to increase the scale-up efficiency of apilot bioprocess. They then examine the impact of
their seed-train intensification strategy on criticalquality attributes of the produced biologic.

Simplifying Intensification

The development and manufacture of biotherapeutics
is a time-consuming, expensive, and often high-riskprocess. Strategies to speed up development andproduction, reduce costs, and promote a robust process
are necessary to remain competitive.
Robust PI solutions from early development tocommercial production help meet industry demands
and create more sustainable, productive, and flexible
facilities. Typically, upstream PI scenarios are thefirst to be explored.
4

BioProcess International

19(10)si

October 2021

N − 1 Perfusion High
Inoculum, Fed-Batch —Batch/MCC Capture

The benefits of seed-train intensification are also
backed up by experimental data. In the following study,a SU production bioreactor was populated with highdensity cultures of CHO cells, facilitating a 30×
reduction in inoculation volume. The intensified
process generated cultures and products withcharacteristics that were comparable to standard fedbatch procedures.
Deciding which strategy to implement can be abarrier to integrating PI in new and existing facilities.
However, following a simplified framework can help
manufacturers tackle the selection process and quickly
reap the benefits of PI philosophies in their facilities.

References

1 Process Intensification: Key Considerations and Expert
Insights. Sartorius, Göttingen, Germany, 2021 (eBook); https://www.sartorius.com/en/723616-723616.
2 Matuszczyk J-C, et al. Intensified Seed Train Strategy
for Faster, Cost-Effective Scale-Up of Biologics
Manufacturing. BioProcess Int. 18(11–12) 2020: 62–64;
https://bioprocessintl.com/sponsored-content/rm-perfusionbioreactors-intensified-seed-train-strategy-for-faster-costeffective-scale-up-of-biologics-manufacturing.
3 Tindal S, et al. Intensifying Upstream Processing:
Implications for Media Management. Sartorius, Göttingen,Germany, 2021 (eBook); https://www.sartorius.com/
en/791518-791518. c
Corresponding author Priyanka Gupta is the head of market entry
strategy for the protein-based therapeutics segment at Sartorius;
priyanka.gupta@sartorius.com, 1-408-872-2442. Katy McLaughlin
is a scientific content writer at Sartorius.

Sponsored

Intensified Seed Train Strategy for
Faster, Cost-Effective Scale-Up of
Biologics Manufacturing
Jens-Christoph Matuszczyk, Johannes Lemke, Markus Schulze,
Sabrina Janoschek, Gerben Zijlstra, and Gerhard GrellerThe 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, economical development of suchtherapies. Process intensification is one approach tohelp shorten manufacturing timelines and reduce
cost of goods (CoG) (1, 2). Today, processintensification in upstream cell culture enables
biologics manufacturing in facilities with smaller
footprints and lower scale-up volumes than was
possible before. Intensified processing of Chinese
hamster ovary (CHO) clones that produce MAbs isbeing developed in the seed train of upstream cell
cultures (3) to support generation of high–celldensity (HCD) cell banks and processes to reduce
plant size, capital investment, and overhead costs
while increasing productivity.
In traditional practice, MAb manufacturing relies onseed ratios of ~1:5 (3) with an initial inoculum of 0.3 ×
106 cells/mL (4) using a seed train of five to eighttransfer steps. Standard seed trains typically begin
with shake flasks in the postthaw expansion (N – 8 toN – 5) and then move through a rocking-motion (RM)
5–50-L bioreactor (N – 4 to N – 3) to seed a series ofN – 2 to N – 1 stirred-tank (STR) bioreactors (200 L and
500 L) ending in a production culture (N) with avolume of 2,000 L. Adopting an intensified cell culture
approach at N – 4 to N – 1 by, for example, using 2D RM
bioreactors in perfusion mode (where cells stay in the
exponential growth phase throughout the entire
culture) could increase viable cell densities to 100 × 106
cells/mL and drive seed ratios of >1:100. That allows
skipping of process-transfer steps. For example, HCD
cell banks can be thawed directly and inoculated into
RM bioreactors at higher volumes (0.5–2 L dependingon the HCD cell-bank volume), thus bypassing cellculture expansion in shake flasks and the potential
errors and contamination issues associated withmanual handling of flasks. Also, with perfusion in theN – 1 seed train, cells can reach higher densities forSponsored

inoculating a pilot or production bioreactor directly
without process transfer through multiple
intermediate-scale STR bioreactors. That reduces boththe number of steps and the total number of seed-train
days, thus reducing initial investment and operational
CoG. However, not all CHO clones that produce MAbs
can be cultured in intensified processing because highcell densities can compromise protein expression levels
and genetic stability (5).
Our goal was to explore the possibility of culturing
a CHO clone that expresses a commercial MAb
therapeutic in HCD for an intensified, scalable cellculture seed-train protocol. We investigated whether
single-use RM perfusion bioreactors (working volume
1–25 L) could achieve a reproducible cell culture withat least 100 × 106/mL viable cell density (VCD) at
different working volumes. The HCD culture wasmonitored using process analytical technology (PAT)at all scales. When the culture in the RM bioreactor(1-L working volume) reached the required HCD, aculture volume was used to seed a pilot-scale STR
single-use bioreactor (200 L) automatically, whichthen ran in fed-batch mode. During the pilot run, we
measured cell density and viability along with the
MAb’s critical quality attributes (CQAs), thencompared those results with the same MAb produced
through a standard seed train. The results established
whether a HCD perfusion seed train can inoculate astandard manufacturing N-stage bioreactor to reduce
the number of transfer steps and whether it is possible
to use a PAT-based control regime for automatedinoculation of N-stage processes to reduce inoculation
volumes.

Materials and Methods

Cell Line: We chose a Cellca CHO-DG44 cell line
expressing a commercially available human MAb toinvestigate the practicality of using HCD perfusion
culture to inoculate a pilot-scale bioreactor. The cellline, with its associated media and cell-culture
parameters, was selected for proof-of-concept studies
October 2021

19(10)si

BioProcess International

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

because the fed-batch process used to produce thisMAb has been well-characterized by Sartorius.
Bioreactors: The Biostat RM 20/50 bioreactor(Sartorius) was chosen for seed-train studies because it
continuously renews the cell-culture medium, enablingmass transfer between the headspace and medium for
efficient, low-shear mixing. The system was used withsingle-use Flexsafe RM perfusion bags (1–25-L working
volume) constructed of pharmaceutical grade lowdensity polyethylene, which is used throughout theentire range of RM and STR bioreactor bags to ensure
consistent quality. The bags each feature an integrated
perfusion membrane at the bottom, forming acompartment for removing cell-free media during theperfusion process for minimal loss of or damage to thecells. The RM perfusion bioreactors scale from 2 L to
50 L (working volume 1 L–25 L) using a common RM
bioreactor design to simplify process transfer.
A Biostat STR bioreactor (200 L) with single-use
Flexsafe STR bags was used as the pilot-scale singleuse bioreactor. The Sartorius STR bioreactor range
scales from 50 L to 2,000 L and uses the same
established bioreactor design principles as stainlesssteel bioreactors. All bags have similar geometries
across scales (5) to facilitate process transfer andscale-up (4).

Proof-of-Concept Studies

Seed Train Study: To demonstrate the performance andscalability of cultures in RM perfusion bioreactor bags,
6

BioProcess International

19(10)si

October 2021

we cultured CHO cells for six days in perfusionbioreactor bags of 2 L, 10 L, 20 L, and 50 L (working
volumes of 1 L, 5 L, 10 L, and 25 L) using proprietary
media and feeds at a minimum cell-specific perfusion
rate (CSPR) of 50 PI (cells/day). The cells were cultured
at 36.8 °C, pH 6.95, and DO 60% (maintained byincreasing the rocking rate at a 10° angle). On-line PAT
sensors, including the BioPAT ViaMass sensor, wereused to monitor and control the culture’s VCD andviability and enable automatic feeding. The 2-L culture
was cultivated for an additional two days under
perfusion conditions to determine a maximum VCD.
Pilot Bioreactor Studies: A VCD model from the RM
perfusion bioreactor was created from the signal fromthe BioPAT ViaMass sensor. With this model, the VCD
was measured on-line constantly, and when cells
reached a trigger point of 100 × 106 cells/mL, a cell
inoculum volume calculated to produce an initial celldensity of 0.33 ± 0.04 × 106 cells/mL was transferred
automatically from the RM perfusion bioreactor to thepilot scale STR bioreactor. Cells cultured in theperfusion bioreactor (400 mL at approximately 100 ×
106 cells/mL) were used to seed a Biostat STR 200-L
single-use bioreactor. Cells were cultured for 12 days
using proprietary media and feeds at 36.8 °C, pH 7.1,
and DO 60%. Feeds were added after a three-day
batch phase on day four. VCD and cell viability data
were measured and collected by the BioPAT ViaMass
sensor. A cell-free supernatant was sampled for
N-glycan profiling to determine MAb product quality
Sponsored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

and titer. The results were compared with historical
data from a standard seed train for which cells were
cultured in standard Biostat RM 20/50 bags (14 L atapproximately 3 × 106 cells/mL) and used to inoculatea Biostat STR 200 L single-use bioreactor.

Results and Discussion

Performance of Seed Train: The results (Figure 1) showthat in six days, the RM perfusion bioreactors cangrow CHO cell cultures with >90% viability and withhigh cell densities of >100 × 106 cells/mL. That
represents a 33-fold increase in VCD compared with an
inoculum from a standard RM bioreactor. Over eightdays, 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 demandcan be supported at such high cell densities.
Our VCD and viability results are consistent at all
scales tested up to 50 L. The reproducibility andscalability of HCD cultures using the perfusionbioreactors shows that this method can produce cellswith high enough VCDs to seed 200-L pilot bioreactors
directly, indicating the potential for inoculating
2,000-L production bioreactors using a suitable HCD
culture, e.g., by using a RM 10 or 20.
Pilot-Scale Study: Cells in proprietary media fromthe RM perfusion bioreactor were used to seed a 200-L
pilot-scale bioreactor using an automated PAT sensorcontrolled inoculation set with a target initial cell
Sponsored

density of 0.33 ± 0.04 × 106 cells/mL. That resulted inan actual starting cell density of 0.37 × 106 cells/mL,
demonstrating that the PAT sensor can achieve
inoculation precision comparable with manual
seeding. In total, 0.4 L of culture from the intensified
seed train was used to inoculate the pilot-scalebioreactor compared with needing over 13 L of cell
culture from a standard seed train. Thus, using HCD
cells to seed the pilot-scale bioreactor required over
30-fold less inoculation volume than when using cells
from a standard seed train.
Fed-batch results (Figure 2) from the pilot-scalebioreactor seeded with HCD cells from the RM
perfusion bioreactor and the traditional fed-batch cell
culture show comparable growth characteristics overthe entire process run. Both achieve a maximum VCD
of 25 × 106 and viability of >90% in seven days. The
MAb titers also were similar, with 3.5 g/L achieved
using the standard seed train compared with 3.2 g/Lfrom the intensified seed train. Those results indicatethat using HCD cells to seed pilot-scale bioreactors has
no adverse effect on cell growth, viability, or titer overa standard 12-day fed-batch run at 200-L scale in asingle-use STR bioreactor.
Product Quality: CQAs of the MAb product from fedbatch cultures inoculated with standard and from
intensified seed trains (Figure 3) showed similar trends
in glycan profiles of G2/G0 and G1/G0 ratios and with
95% fucosylated forms produced. The performance
October 2021

19(10)si

BioProcess International

7

4.5

90

4.0

80

3.5

70

3.0

60

2.0

1.0
0.5
0.0

G1/G0 ratio

1.5

G2/G0 ratio

STR Fed-Batch Standard Seed

results of the cells cultured in the pilot-scale bioreactor
from the intensified seed train show that using HCD
cells to seed a pilot-scale bioreactor does not affectproduct quality. That indicates the usefulness of thisapproach for a hybrid perfusion/fed-batch processscale-up.

Productivity and Quality

The proof-of-concept studies presented here show thatsingle-use RM perfusion bioreactors 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 (inoculated bymeans of automated process control) and cells from astandard fed-batch culture used to seed a 200-L STR
bioreactor produce comparable cell growth, viability,
titer, and MAb CQA profiles. Our results indicate thatusing this intensified seed-train approach canincrease productivity without compromising productquality and could potentially increase the costeffectiveness of biologics production.

References

1 Anderlei T, et al. Facility of the Future. DECHEMA
Report 2018; https://dechema.de/dechema_media/
Downloads/Positionspapiere/SingleUse_FoF+2018+engl.pdf.
2 BioPhorum Operations Group Report.
Biomanufacturing Technology Roadmap 2017: Process
8

BioProcess International

19(10)si

October 2021

G1/G0 ratio

2.5

Fucosylated forms (%)

100

Fucosylated forms (%)

5.0

50
40

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 from standard or intensified seed trains

30
20

G2/G0 ratio

STR Fed-Batch Intensified Seed

100

Technologies; https://www.biophorum.com/download/
process-technologies.
3 Wittmann C, et al. 2016 Industrial Biotechnology:
Products and Processes. Wiley VCH Verlag GmbH & Co.
KGaA: Weinheim, Germany, 2016; https://doi.
org/10.1002/9783527807833.
4 Matuszczyk J-C, et al. A Rapid, Low-Risk Approach forProcess Transfer of Biologics from Development to
Manufacturing Scale. BioProcess Int. 18(5) (2020): 44–51;
https://bioprocessintl.com/sponsored-content/biostat-strbioreactors-a-rapid-low-risk-approach-process-transfer-ofbiologics-from-development-to-manufacturing-scale.
5 Rees-Manley A. Evaluation of a Small-Scale Perfusion
Mimic for Intensified Processes. Gen. Eng. News 2019;https://www.genengnews.com/resources/tutorial/evaluationof-a-small-scale-perfusion-mimic-for-intensified-processes.
6 Noack U, et al. Single-Use Stirred Tank Reactor
BIOSTAT CultiBag STR: Characterization and Applications.
Single-Use Technology in Biopharmaceutical Manufacture.
Eibl R, Eibl D (Eds). Wiley: Hoboken, NJ, 2010: 225–240;
https://doi.org/10.1002/9780470909997.ch19. c
Corresponding author Jens-Christoph Matuszczyk is manager,
bioprocessing upstream, Sartorius, Göttingen, Germany; 49-551308-4245; Jens.Matuszczyk@sartorius.com. Johannes Lemke is ascientist and Markus Schulze and Sabrina Janoschek are PhD
students. Gerben Zijlstra is a global technology consultant, andGerhard Greller is a senior scientist at Sartorius Stedim Biotech
GmbH. Editorial contact: Avani Jain, Sartorius; avani.jain@
sartorius.com This article is reprinted from BioProcess International
18(11−12) 2020.
Sponsored