C ontinuous P rocessing TECHNOLOGIES

A Look At Perfusion
The Upstream Continuous Process

LY
Cynthia Challener, PhD

N
O
A
lthough implementation of Figure 1  Perfusion culture process

N
continuous manufacturing for
biopharmaceuticals is in the Balance
early stages, continuous cell Pump

IO
culture technology has been around XRS Bioreactor
System
for close to thirty years. Perfusion was

S
initially developed in the late 1980s as
a means for increasing protein titers Pump
Balance

IS
(1). However, high costs driven by
media consumption limited
widespread commercial adoption. In
M
Medium Supply
the same time frame, advances in cell
line engineering, media composition,
Direction of Medium Flow
R

and bioreactor design led to 10-fold

increases in titers for batch and
E

fed-batch modes, eliminating the first

driver for adoption of perfusion
P

Perfusate
technology. As a result, over the next
20 years or so, perfusion processes
were largely used only for production
H

of toxic or unstable proteins that tangential-flow filtration (TFF). efficient facility use. As a result,
degrade if exposed to culture Another option is to retain the cells by perfusion processes are an attractive
IT

conditions for typical batch/fed-batch binding them to a substrate (capillary solution for flexible, single-use,
residence times. Recently, however, fibers, membranes, microcarriers in multiproduct manufacturing facilities.
W

growing recognition in the industry fixed bed, and so on) in the bioreactor. Continuous operation in smaller
that there is a need for alternative Other methods include use of equipment also has the potential to
manufacturing strategies that can centrifuges. Lately, a revival of eliminate the need for extensive
T

boost efficiency and productivity methods using acoustic waves has process scale-up and technology
while reducing costs has led to been seen (2). transfer.
IN

renewed interest in perfusion Continuous removal of a biologic

technology. Many Potential Benefits drug substance from the bioreactor is a
One of the biggest advantages of second important advantage of
R

What Is Perfusion Cell Culture? perfusion cell culture is the ability to perfusion cell culture. Many proteins
A perfusion cell culture process achieve optimum, steady-state are unstable under batch/fed-batch
P

involves the constant feeding of fresh conditions, which can lead to conditions as toxic byproducts build
media and removal of spent media and significantly higher productivities up during a run. Degradation and
E

product while retaining high numbers (grams/liter of bioreactor working undesired modification are minimized
R

of viable cells (Figure 1). Removing volume per day). Smaller scale single- because the time a product spends in
spent media while keeping cells in use bioreactors can thus be used, the bioreactor in perfusion mode is
culture can be done using alternating leading to reduced capital and limited. Steady-state operation also
tangential-flow (ATF) and standard operating costs combined with more leads to more consistent product

44 BioProcess International 14(6)s J une 2016 Sponsored Supplement

 Pall Solutions That Support Perfusion Processes
Continuous cell culture requires a bioreactor design that can
support long-term steady-state performance and enable ease of
product recovery. The iCELLis bioreactor from Pall — the first
fully-integrated, single-use perfusion bioreactor with disposable,
preinstalled calibrated probes and needing no additional
oxygen device — combines the advantages of single-use
technologies with the benefits of a fixed-bed system, providing
the perfect solution for process intensification.
iCELLis provides a large-cell-growth surface area in a very small
volume, accommodating up to 500 m2 of growth area in a
single-use cassette measuring 38 inches in diameter by 12
inches high. Just as important, because adherent or suspension
cells are immobilized or entrapped in the fixed-bed and the
iCELLis system operates in perfusion mode, product harvesting
and further downstream processing are simplified. Last but not
least, to reduce process development time, the perfusion device
is integrated into the iCELLis skid.
An iCELLis fixed-bed filled with custom carriers can dramatically
simplify cell culture processes. A unique waterfall oxygenation
system is complemented by gentle agitation and biomass
immobilization to enable high-cell densities that equal the
productivity of much larger stirred tank units; higher specific
productivities can also be achieved. The growth matrix inside
the iCELLis system is a favored growth environment that,
together with controlled cell culture parameters, can
significantly increase biomass amplification and maintain
specific productivity. In addition, little adaption of protocols is
required when switching to an iCELLis bioreactor, enabling easy,
straightforward scale-up for the majority of mammalian cell
lines.
The iCELLis Nano system is ideal for feasibility studies and small-
scale production, whereas the iCELLis 500 system is designed for
industrial-scale manufacturing up to 500 m2. It is important to
note that the bioreactor exhibits linear scalability from R&D to
manufacturing.
Harvesting of perfusion bioprocesses can also be a challenge. To
facilitate fully integrated cell culture and harvesting operations,
Pall has developed its unique Cadence Acoustic Separation
Technology, cutting-edge acoustic wave separation technology
developed by FloDesign Sonics (FDS) and exclusively licensed by
Pall.
With the Cadence acoustic separation system, continuous
removal of cells is achieved in a closed system without
centrifugation or filtration, thus streamlining this challenging
step in the biologics manufacturing process. Compared to large-
scale depth filtration, the Cadence Acoustic Separation system
allows for the reduction of 75% of the required buffer volume,
leading to significant cost savings and a reduction in tank sizes
and overall operational footprint.
Furthermore, the Cadence Acoustic Separation system is
effective for clarifying different types of recombinant
therapeutic proteins and monoclonal antibodies, regardless of
variations in particulate concentration and cell culture density,
turbidity and viability.
In addition to facilitating continuous processing, both the
iCELLis bioreactor and Cadence Acoustic Separation system help
reduce capital expenditures and operating costs.

quality, which can lead to simpler
downstream purification needs.
No Widespread Use — Yet
Despite these advantages, perfusion is
not yet widely used for
biopharmaceutical manufacturing.
Although most drug companies are
exploring the potential benefits of
perfusion at process development and
clinical scales, only a few companies
use the technology on a regular basis.
It remains to be demonstrated whether
parameters that directly influence
product quality (such as protein
folding, aggregation, glycosylation,
oxidation, and contamination) have
yet to be developed.
Resistance to change — an issue
when switching any unit operation
from batch to continuous mode —
also must be overcome before
perfusion technology becomes the cell
culture mode of choice.
Role of Equipment Suppliers
robust for long-term operation and are
scalable, cost-efficient, and easy to
use, while producing higher quality
end products in less time.
References
1 Bonham-Carter J. A Brief History of
Perfusion Biomanufacturing, BioProcess Intl.
9(9) 2011; www.bioprocessintl.com/upstream-
processing/bioreactors/a-brief-history-of-
perfusion-biomanufacturing-322322.
2 Shirgaonkar IZ, Lanthier S, Kamen A.
Acoustic Cell Filter: A Proven Cell Retention
Technology for Perfusion of Animal Cell

lower costs and accelerated
development times can be achieved
using perfusion.
In addition, integration of
perfusion cell culture processes with
continuous harvesting and other
continuous downstream unit
operations has only just begun to be
explored. Although there have been
advances in sensor technology for real-
time monitoring of basic process
conditions, process analytical
technology for on-line monitoring of
more complex critical process
www.ncbi.nlm.nih.gov/pubmed/15135491. •
Cultures. Biotechnol Adv. 22(6) 2004: 433–444;
Vendors such as Pall have a key role to
play in facilitating adoption of
continuous biopharmaceutical
Cynthia Challener, PhD, is a freelance
manufacturing and can guide
technical writer, editor, and market
customers as they seek to integrate researcher for the chemical and allied
perfusion as an enabling technology. industries; www.pall.com/continuous-
As the team continues to hone questions.
expertise and gain knowledge, Pall
sees a future where advanced
technologies can be applied in a novel
way, starting with perfusion. The
ultimate goal remains to provide
customers with bioprocess
technologies that are sufficiently

Sponsored Supplement J une 2016 14(6)s BioProcess International 45