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Received: 22 March 2018 | Revised: 24 July 2018 | Accepted: 30 July 2018

DOI: 10.1002/bit.26812

REVIEW

Progression of continuous downstream processing of

monoclonal antibodies: Current trends and challenges

Balaji Somasundaram1 | Kristina Pleitt1 | Evan Shave1,2 | Kym Baker2 |

1,3
Linda H. L. Lua

1
Australian Research Council Training Centre
for Biopharmaceutical Innovation, Australian Abstract
Institute for Bioengineering and Rapid advances in intensifying upstream processes for biologics production have left
Nanotechnology, The University of
Queensland, Brisbane, Queensland, Australia downstream processing as a bottleneck in the manufacturing scheme. Biomanufacturers
2
Patheon Biologics—a part of Thermo Fisher are pursuing continuous downstream process development to increase efficiency and
Scientific, Brisbane, Queensland, Australia
flexibility, reduce footprint and cost of goods, and improve product consistency and
3
Protein Expression Facility, The University of
Queensland, Brisbane, Queensland, Australia quality. Even after successful laboratory trials, the implementation of a continuous
process at manufacturing scale is not easy to achieve. This paper reviews specific
Correspondence
Linda H. L. Lua, Protein Expression Facility, challenges in converting each downstream unit operation to a continuous mode. Key
The University of Queensland, Brisbane, elements of developing practical strategies for overcoming these challenges are detailed.
Queensland 4072, Australia.
Email: l.lua@uq.edu.au These include equipment valve complexity, favorable column aspect ratio, protein‐A resin
selection, quantitative assessment of chromatogram peak size and shape, holistic process
Funding information
Australian Research Council, Grant/Award characterization approach, and a customized process economic evaluation. Overall, this
Number: IC160100027 study provides a comprehensive review of current trends and the path forward for
implementing continuous downstream processing at the manufacturing scale.

KEYWORDS
continuous manufacturing, continuous chromatography, process scale‐up and membrane
adsorbers

1 | INTRODUCTION 20 biopharma companies are expected to invest $109 billion in

The global biopharmaceutical market is growing at a compound annual
rate of 10% since 2014 and is expected to reach approximately $390
billion by the end of 2019 (Ayturk & Marshall, 2016). Therapeutic
monoclonal antibodies (mAb) are a key class of biopharmaceutical
products that has grown briskly in product approvals and sales, from
the time when the first mAb was commercialized (Ecker, Jones, &
®
Levine, 2015). As shown in Figure 1a, Humira , an antitumor necrosis
factor‐α mAb manufactured by AbbVie (AbbVie Inc., North Chicago, IL),
achieved gross sales of $12.5 billion in 2014 (Udpa & Million, 2016) and
increased by 28.8% in 2016 to reach $16 billion (Lindsley, 2017). The
biopharmaceutical market has become more dynamic with the recent
increase in the entry of biopharma companies into the biosimilars
business (Udpa & Million, 2016). To maintain a competitive space in
such a rapidly expanding market, in the next 5 years, the top
research and development (R&D; Figure 1b,c) and the overall R&D
expenditure is expected to increase 2.8% each year to reach $182
billion by 2022 (EvaluatePharma, 2016). From a process development
perspective, most of the R&D is focused on intensifying and integrating
bioprocess technologies to increase efficiency and flexibility in
biopharmaceutical manufacturing (Croughan, Konstantinov, & Cooney,
2015; Estes & Longer, 2017). In this quest for faster and more efficient
processes, continuous manufacturing, designed to enhance efficiency,
has emerged as a new modality in biomanufacturing.
This study elaborates on the current trends and potential risks in
continuous downstream processing of mAbs with a primary focus on
continuous chromatography. We provide insights into the ways to
move from bench‐scale solutions to industrial implementation.
Furthermore, the article highlights the importance of a holistic
approach to process development and characterization to address

Biotechnology and Bioengineering. 2018;115:2893–2907. wileyonlinelibrary.com/journal/bit © 2018 Wiley Periodicals, Inc. | 2893

2894 | SOMASUNDARAM
F I G U R E 1 (a) 2014–2016 gross annual sales of products for
which biosimilars are in development (source: EvaluatePharma,
2016, Lindsley, 2017; Udpa & Million, 2016). (b) A 5‐year forecast
of R&D expenditure for the top 20 biopharma companies,
(e.g., 2016–2021; source: EvaluatePharma, 2016). (c) A 5‐year
forecast of overall R&D expenditure for the biopharma companies
(source: EvaluatePharma, 2016)
the regulatory challenges related to introducing continuous down-
stream processing at manufacturing scale.
2 | EVOLVING TRENDS IN UPSTREAM
PROC ESSIN G
Upstream process development for mAb production typically
involves (a) clonal cell line selection and (b) media and bioreactor
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ET AL.
conditions optimization (Li, Lee, Zhou, Tressel, & Yang, 2006). High‐
throughput clone screening and selection using fluorescence‐acti-
vated cell sorting along with the implementation of miniaturized
bioreactors to optimize media, feeding regime and bioreactor
production has decreased the timeframe for cell culture process
development (Li et al., 2006). Rameez, Mostafa, Miller, and Shukla
(2014) have demonstrated the use of 10–15 ml advanced microscale
bioreactors (ambr™, TAP Biosystems, part of the Sartorius Stedim,
Greenville, Delaware) to optimize expression process parameters for
achieving optimum cell growth, viability, and product titer. The cell
culture profiles achieved in this microscale bioreactor study was
consistent with 3, 15, and 200 L stirred‐tank bioreactors.
For large‐scale production, biologics manufacturers either take a
fed‐batch approach for the ease of implementation (Lim, Sinclair,
Shevitz, & Bonham‐Carter, 2011), a perfusion process for increased
productivity (Kunert & Reinhart, 2016), or a combination of perfusion
and fed‐batch (W. C. Yang et al., 2014). Under constant supply of
fresh nutrients in a perfusion culture with enhanced cell densities,
mAb productivity reached 425 mg/L/day after 12 days, while over
the same period fed‐batch produced 55 mg/L/day (Langer & Rader,
2014). The evolution of continuous perfusion bioreactors and the
availability of established single‐use upstream technologies offer cost
and performance benefits to biologics manufacturers. A recent cost
of goods (CoGs) evaluation reported 30% savings can be achieved in
an integrated continuous process using disposable technologies over
a stainless steel batch process (Hummel et al., 2018). The upstream
process has also significantly advanced in the development of
process analytical tools for monitoring bioreactor performance,
meeting the requirements of the Food and Drug Administration
(FDA) for the implementation of continuous manufacturing. Various
in‐process, on‐line, and at‐line analytical tools used during upstream
mAb production have been reviewed in detail (Li, Vijayasankaran,
Shen, Kiss, & Amanullah, 2010). While the industry has witnessed
major developments in upstream processes, there has been little
progress in downstream processing. This has resulted in the
continuous accumulation of an increased amount of product (Clincke
et al., 2013) thereby making downstream processing a “bottleneck”
in the manufacturing scheme (Gottschalk, 2008).
3 | E V O L V I N G T R E N D S I N M ID S T R E A M
PROCESSING
Continuous centrifugation, depth filtration or tangential flow filtra-
tion (TFF) have been used as primary clarification techniques at
manufacturing scale to remove the bulk of large particles, cell debris,
and whole cell impurities (Supporting Information Figure S1a;
Berthold & Kempken, 1994). Continuous centrifugation, operating
at 10,000 to 12,000g with a short residence time, is not highly
efficient at removing particles <1 µm (Yavorsky, Blanck, Lambalot, &
Brunkow, 2003). Moreover, at high cell densities a centrifugation
process requires frequent desludging, leading to a decrease in
product recovery (Popova, Stonier, Pain, Titchener‐Hooker, & Farid,
 T A B L E 1 Comparison of common clarification technologies (based on manufacturer data)

Feed stream
Acoustic wave Alternating tangential Continuous counter pretreatment with
Parameter separation flow filtration Continuous centrifugation flow centrifugation Depth filtration depth filtration
SOMASUNDARAM

Optimized total cell 20–50 20–146 3–13 6–150 10–30 >30

ET AL.

density at time of
clarification
(×106 cells/ml)a
Typical viability at time >60 >70 >40 >80 >40 >40
of clarification (%)a
Typical culture volume (L) 3–2,000 1–1,000 80–5,000b 0.1–6,000 0.1–2,000b 0.1–5,000b
Scalability Yes Yes Noc Semic Yes Yes
d
Continuous processing Yes Yes Yes Yesd No No
feasibility
References Collins and Clincke et al. Berthold and Kempken (1994), Masri (2017) Tomic et al. (2015)
Levison (2016) (2013) and Warikoo and Yavorsky et al. (2003)
et al. (2012)
a
Conditions at the time of harvest and operating parameters are process dependent and should be determined during the process development.
b
Technology is scalable beyond listed upper limit. Scale restrictions are based on infrastructure and when it becomes impractical from an economic standpoint.
c
While centrifugation has been used with culture volumes greater than 2,000 L, performance at small scale is not necessarily predictive of performance at large scale.
d
Operation lends itself to continuous processing, but auxiliary activities (e.g., assembly, disassembly, and cleaning) may reduce the benefits of continuous operation.
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2896 | SOMASUNDARAM
2016). This limits the use of continuous centrifugation as
a standalone unit operation for clarification and hence requires a
secondary clarification technique like depth filtration (Berthold &
Kempken, 1994). Similarly, single stage clarification is not feasible
with depth filters or TFF. In a manufacturing set‐up, cleaning,
steaming, cooling, assembly, and disassembly of clarification hard-
ware is a significant rate limiting step (Yavorsky et al., 2003) and can
potentially nullify the productivity advantages of an integrated
continuous process. Alternatively, clarification process with single‐
use technology (SUT) using presterilized single‐use filters can
increase process efficiency by eliminating cleaning, sterilization, and
testing steps (Whitford, 2014).
Alternating tangential flow filtration (ATF) cell retention system
(Warikoo et al., 2012), acoustic wave separation (AWS) technology
(Collins & Levison, 2016), continuous counter flow centrifugation
(Masri, 2017), and pretreatment of cell culture harvest with a
polycationic flocculating agent (e.g., pDADMAC; Burgstaller et al.,
2017; Tomic et al., 2015) are other clarification technologies
(Table 1). A 3–10‐fold reduction in secondary depth filter area, with a
throughput of 15 L·m−2·h−1, was achieved by using AWS technology as
the primary clarification operation (Supporting Information Figure S1b;
Collins & Levison, 2016). Depth filtration of pDADMAC‐flocculated
broths had a 4‐fold reduction in filter area when compared to traditional
two‐stage depth filtration (Burgstaller et al., 2017; Tomic et al., 2015).
Using a tubular reactor, an online focus beam reflectance measurement
and inline pressure sensor‐controlled valves, Burgstaller et al. (2017)
demonstrated that depth filtration of flocculated cell culture could be
operated in a continuous manner. While flocculating agents are
beneficial in reducing depth filter area, demonstrating flocculant
clearance in the process intermediates using subsequent unit operations
is a challenge. Warikoo et al. (2012) have demonstrated the use of ATF
by directly loading the ATF clarified material onto the periodic counter‐
current (PCC) system without additional clarification (Supporting
Information Figure S1c). These technologies have shown to reduce or
bypass the need for secondary depth filtration, thereby decreasing
operational footprint.
Efficient clarification of cell culture harvest depends on a
combination of both total cell density and viability (Tomic et al.,
2015). AWS and flocculation technologies demonstrate capabilities of
handling high cell densities at a range of viabilities (25–50 × 106 cells/
ml at 60% viability and >30 × 106 cells/ml at 35–80% viability,
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ET AL.
respectively; Table 1). Among all available technologies, ATF and
continuous counter flow centrifugation can handle cell densities of
over 100 × 106 cells/ml, making them well suited for harvesting high
cell density continuous perfusion processes (Table 1). The continuous
counter flow centrifugation is a single use technology with 100 ml to
6 L chamber capacity (Masri, 2017). Such a wide range of operational
flexibility is an advantage during continuous integrated process
development. Nevertheless, economic analysis on COGs and harvest
time for manufacturing scale bioreactors (e.g., 20,000 L production) is
critical for this new technology, particularly for less‐stable proteins.
Among all midstream process technologies, ATF and TFF have
demonstrated capabilities of integrating with perfusion reactor and
Protein‐A (ProA) chromatography in a continuous mode, while
technologies like the AWS and depth filtration are used to clarify
material from fed‐batch bioreactors to feed the continuous ProA
chromatography step.
4 | D O W N S T R E A M P L A T F O R M P RO C E S S
A mAb purification platform involving two to three chromatographic
and filtration steps (Figure 2; Fahrner et al., 2001; Low, O’Leary, &
Pujar, 2007) typically accounts for 50–80% of the production costs of
therapeutics (Farid, 2007; Gavara, Bibi, Sanchez, Grasselli, &
Fernandez‐Lahore, 2015). The process suffers from the under-
utilization of the expensive ProA column to avoid breakthrough
(Mahajan, George, & Wolk, 2012) and biomanufacturing facilities
often trade‐off between production rate and operational costs by
increasing the number of ProA cycles (typically 3–5 times) to reduce
the column volume (Low et al., 2007; A. A. Shukla, Hubbard, Tressel,
Guhan, & Low, 2007). With the need to balance, the cost of raw
materials against production costs, organizational design (shift
management) and commercial demand, biomanufacturing facilities
have come under pressure to improve the efficiency of their
downstream manufacturing process. For this purpose, integrated
continuous downstream processing is considered as a potential
solution to reduce operational costs, increase efficiency and
flexibility, streamline processes, reduce footprint, and improve
product consistency and quality (Bisschops & Brower, 2013; Gjoka,
Gantier, & Schofield, 2017; Hernandez, 2015; Jacquemart et al.,
2016; Warikoo et al., 2012).
F I G U R E 2 A typical mAb purification
platform process involving three
chromatography unit operations

SOMASUNDARAM ET AL.
5 | CONT IN UOUS DOW NSTREAM
PROC ESSIN G
Continuous chromatography utilizes multiple smaller columns to
maximize the usage of resin’s binding capacity (Figure 3a). The use of
smaller columns provides an economic advantage by reducing
purification suite footprint, buffer and resin usage (Godawat et al.,
2012). Increase in resin capacity coupled with shorter residence time
can potentially improve productivity (g/Lresin/day; Hernandez, 2015;
Mothes, Pezzini, & Schroeder‐Tittmann, 2016; Nestola et al., 2015;
Schaber et al., 2011). Moreover, continuous chromatography is
suited for purification of less‐stable recombinant proteins, as the
reduced process time helps ensure stability of the protein. The
continuous chromatography process development has progressed
tremendously in recent years due the availability of several
commercial continuous chromatography systems (Table 2).
F I G U R E 3 Schematic representation of
downstream process intensification.
(a) Downstream processing involving
multicolumn continuous chromatography
within each unit operation.
(b) Downstream processing scheme
showing all unit operations performed
simultaneously and continuously.
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| 2897
The first successful integration of a perfusion bioreactor and
continuous protein capture using a GE Healthcare AKTA pcc 75 four‐
column PCC system (Uppsala, Sweden) was demonstrated in
Warikoo et al. (2012). This study explored the operational flexibility
and process train simplification of downstream process by removing
intermediate hold steps. In 2014, Genzyme Corporation patented an
integrated and continuous process for manufacturing a therapeutic
protein drug substance (DS). The patented process included
continuous ProA capture, viral inactivation, and continuous polishing
using two multicolumn chromatography systems. This integrated
continuous production process delivered DS with consistent product
critical quality attributes (CQA; Konstantinov, Godawat, Warikoo, &
Jain, 2017). In the following year, an uninterrupted and fully
automated end‐to‐end continuous purification of antibody DS with
a process productivity of >600 g/L resin/day was demonstrated by
Godawat, Konstantinov, Rohani, and Warikoo (2015). Taking a step
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2898 | SOMASUNDARAM ET AL.

T A B L E 2 Comparison of commercial continuous chromatography system specification

Maximum operating Maximum number

Manufacturer Purification system Flow rate pressure (MPa) of columns Operating platform

Pall Life Science Cadence™ BioSMB PD Up to 70 or 1 16 Simulated moving bed

system 700 ml/mina
Cadence™ BioSMB 5–80 L/h 0.4 8
Process 80 system
Cadence™ BioSMB 30–350 L/h 0.4 8
Process 350 system
GE Healthcare AKTA™ pcc 75 1–75 ml/min 2 4 Periodic countercurrent
chromatography
BioProcess™ 1–60 L/h 2 4
PCC 4.7 mm
BioProcess™ PCC 6 mm 4–180 L/h 2 4
BioProcess™ PCC 10 mm 15–600 L/h 2 4
Novasepc BioSC® Lab Scale 1–30 ml/min 0.6 6 Sequential chromatography
®
BioSC Pilot Scale 30–1,500 ml/min 0.6 6
b
Semba Biosciences Semba ProPD™ Up to 70 or 100 1.86 8 Multicolumn continuous
or 300 ml/mina chromatography
ChromaCon Contichrom CUBE Up to 36 or 5 2 Periodic countercurrent
100 ml/mina chromatography
EcoPrime Twin GMP Up to 0.6 or 2.4 0.75 2
system or 9 L/mina
a
The flow rate depends on the pump selection.
b
Semba ProGMP™ system capable of processing 2,000 L is available (product specification to be released).
c
Customized commercial scale system.

further from the DS, the Novartis‐MIT Centre for Continuous
Manufacturing is focusing on continuous manufacturing of the final
vialled drug product (Schaber et al., 2011).
In another study, an integrated downstream bioprocess deliver-
ing purified mAb product was demonstrated using a 25 L fed‐batch
bioreactor (Gjoka et al., 2017). An eight‐column ProA (5‐cm bed
height) process at a linear flow velocity of 360 cm/hr was used to
capture the mAbs. Following viral inactivation, the ProA eluate was
further purified by an anion exchange membrane and mixed‐mode
cation exchange resin. The process was performed in an integrated
continuous mode over 30 cycles with 4.5 log reduction in host cell
proteins and 50% decrease in high molecular weight species (<2%).
Compared with a batch process, the integrated continuous 25‐L
process showed superior economic advantage by utilizing 96% less
ProA resin, 74% less anion exchange membrane, 97% less mixed‐
mode cation exchange resin, and 44% less process buffer. This offers
a COGs advantage for high‐cost processes.
As an alternative, an accelerated seamless antibody purification
approach was demonstrated by Sanofi (Paris, France; Mothes et al.,
2016). In this process, all chromatography unit operations were
performed in a continuous mode with only four buffers, eliminating
intermediate hold times (Figure 3b). As several batch processes are
carried out simultaneously, the time required to convert a batch
process to continuous mode is minimal and the industry can define
and identify a unique batch more readily for regulatory purposes.
Regardless of the economic benefits of operation in a continuous
mode, some limitations such as complexity in valve arrangements,
packing variability with multicolumn systems, operating robustness,
challenges in process characterization, validation and regulatory
concerns hold their implementation at larger manufacturing scales
(Mothes et al., 2016; Stock, Bisschops, & Ransohoff, 2014).
5.1 | Continuous protein A capture
ProA capture typically accounts for 25% (Costioli, Guillemot‐Potelle,
Mitchell‐Logean, & Broly, 2010) of total downstream cost. Supporting
Information Figure S2 shows a breakdown of ProA capture cost,
where consumables (e.g., resin) account for 67% of the cost. To offset
the cost associated with ProA resin and to maximize the advantage of
a continuous process, it is important to select a suitable ProA resin
that has the ability to operate at a faster flow rate while retaining
high binding capacity and longer lifetime (Schaber et al., 2011).
The first step in designing a ProA purification process is to select
an appropriate resin. Table 3 provides a comparison of characteristics
for several commercially available ProA resins. In general, higher
dynamic binding capacity (DBC) is observed at a longer residence time
(RT). DBC is a measure of the amount of target protein the
chromatographic media can bind under respective flow conditions
before a significant amount of target protein breakthrough (Do et al.,
2008). Hence, DBC is as an important parameter reflecting mass
 T A B L E 3 Comparison of protein A affinity media properties (based on manufacturer datasheets)

Maximum reported
Particle size DBC (mg human Residence
Manufacturer Resin name Base matrix (d50v; µm) IgG/mlresin) time (min) Ligand type Ligand attachment
SOMASUNDARAM

BioRad UNOsphere SUPrA™ Highly cross‐linked polymer 53–61 23–27 2 Recombinant protein A Epoxy
ET AL.

GE Healthcare MabSelect™ Highly cross‐linked agarose 85 30 2.4 Recombinant protein A Epoxy (single point)

MabSelect Xtra™ Highly cross‐linked agarose 75 40 2.4 Recombinant protein A Epoxy

MabSelect SuRe™ Highly cross‐linked agarose 85 35 2.4 Alkali‐stabilized protein Epoxy (single point)
A‐derived

MabSelect SuRe™ LX Highly cross‐linked agarose 85 60 6 Alkali‐stabilized protein Epoxy (single point)
A‐derived

MabSelect™ PrismA Highly cross‐linked agarose 60 80 6 Alkali‐stabilized Protein Epoxy (single point)
A‐derived

MabSelect SuRe™ pcc Highly cross‐linked agarose 50 60 2.4 Alkali‐stabilized protein Epoxy (single point)
A‐derived
Merck ProSep®‐vA High Capacity Controlled pore glass 74–125 >20 3–6 Native protein A NG
ProSep®‐vA Ultra Controlled pore glass 75–125 35 2.4 Native protein A (Multipoint)
®
ProSep Ultra Plus Controlled pore glass 60 >50 3–6 Recombinant structurally NG
conserved (native) protein A
Eshmuno A Hydrophilic polyvinyl ether 50 40–55 3–6 Proprietary ligand derived NG
from the C domain of S. aureus
Tosoh TOYOPEARL® AF‐rProtein Hydroxylated methacrylic 45 30 3 Alkali‐stabilized protein A (Multipoint)
A‐650F polymer

TOYOPEARL® AF‐rProtein A Hydroxylated methacrylic 45 65a 5a Alkali‐stabilized protein A (Multipoint)

HC‐650F polymer
Thermo Fisher POROS™ MabCapture™ Cross‐linked poly(styrene 45 ≥37 4 Recombinant native protein A Covalent
Scientific A divinylbenzene) immobilization
POROS™ MabCapture™ Cross‐linked poly(styrene 45 ≥37 4 Recombinant native protein A Covalent
A Select divinylbenzene) immobilization

Note. DBC: dynamic binding capacity; NG: not given .

a
At a 2‐min residence time, the binding capacity is 50 mg human IgG/mlresin.
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2900 | SOMASUNDARAM
transfer limitations that may occur with the increase in flow rate.
Apart from flow conditions, other major differences in the base matrix
type, particle size, ProA ligand density, modifications, and attachments
observed across different resins is also expected to influence the DBC
(Hahn et al., 2005; Lain, 2013). Higher mass transfer rates are
expected in resins with smaller particle diameter (Hahn, Schlegel, &
Jungbauer, 2003; Hahn et al., 2003). For instance, MabSelect Sure™
pcc (GE Healthcare, Uppsala, Sweden) (50‐µm particle) and TOYO-
PEARL® AF‐rProtein A HC‐650F (Tosoh Bioscience, Tokyo, Japan)
(45 µm particle) demonstrate high DBCs at shorter RT (60 mg/mlresin
at 2.4 min and 50 mg/mlresin at 2 min, respectively). The base beads of
both resins are suitable for high mass transfer rates, but porous and
rigid enough to accommodate faster flow rates with minimal pressure
drop across the chromatography bed.
The second important factor in selecting a ProA resin for
continuous processing is the resin reusability. A number of factors
including operating conditions such as, CIP regime, feed stream, and
elution conditions that affect ligand stability, will impact resin
lifetime (Jiang, Liu, Rubacha, & Shukla, 2009; Liu, Ma, Winter, &
Bayer, 2010; A. S. Rathore & Sofer, 2012). As shown in Table 3 and
Supporting Information Table S1, there are a variety of ligands and
recommended CIP strategies based on ligand and base bead stability.
Due to the common availability and demonstrated performance at
reducing bioburden, sodium hydroxide tends to be the preferred
method for cleaning (Biosciences, 2001; Burgoyne, Priest, Roche, &
Vella, 1993; Girot, Moroux, Duteil, Nguyen, & Boschetti, 1990). As a
result, many resins have alkali‐stabilized ProA ligands. Even with
these modifications, repeated exposure to cell culture harvest and
sanitization solutions will degrade ProA resin, resulting in leached
ProA (Carter‐Franklin, Victa, McDonald, & Fahrner, 2007) and
decreased binding capacity over time, leading to an increase in
protein breakthrough (Hober, Nord, & Linhult, 2007). Leached ProA
ligand is a concern as studies have demonstrated it may cause
immunogenic responses (Gómez et al., 2004). However, most of the
vendors have demonstrated that their resins will retain the majority
of the binding capacity with the recommended CIP conditions for
over 100 cycles and subsequent downstream unit operations are
known to clear residual ProA leachates (Lain, 2013; Thillaivinayaga-
lingam, Reidy, Lindeberg, & Newcombe, 2012). Therefore, taking
DBC, RT, particle size, and resin lifetime into consideration,
MabSelect Sure™ PCC and TOYOPEARL® AF‐rProteinA HC‐650F
will be suitable resins for continuous processing requiring fast mass
transfer, without compromising on binding capacity.
5.2 | Continuous viral clearance
Demonstrating virus safety is an important regulatory requirement
for biologics expressed in human or animal cell lines (FDA, 1998).
Viral contamination of biologics, having the potential to impact
patient safety, can occur due to the source of cell line or due to the
introduction of adventitious agents during the manufacturing process
(Aranha, 2011). Currently, adventitious agents are tested via qPCR
assays, animal infection susceptibility, immune responses, or cell
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ET AL.
culture infectivity assays (Gombold et al., 2014; Johnson, Brown,
Lute, & Brorson, 2017). From an upstream perspective, continuous
perfusion mode bioreactors that run for months in comparison to
fed‐batch bioreactors, requires more vigilance to ensure that novel
infectious contaminations do not occur. Raw material sourcing, virus/
mycoplasma filtration of cell culture media and new characterization
techniques have to be considered to reduce the risk of viral
contamination in the upstream stage. Failure to mitigate the risk of
viral contamination can lead to consequences such as facility
shutdown and decontamination of manufacturing facilities, for
example, Genzyme’s production of Cerezyme® and Fabrazyme®
was stopped (from 2009 to 2010 and 2012, respectively) due to the
detection of a Vesivirus 2117 contamination at cell culture stage
(Bestwick & Colton, 2009; Qiu et al., 2013).
Traditionally, in mAb downstream processes, viral clearance is
achieved by a combination of techniques including chromatographic
separations, viral inactivation, and viral filtration methods (Connell‐
Crowley et al., 2012; Farshid, Taffs, Scott, Asher, & Brorson, 2005;
Miesegaes, Lute, & Brorson, 2010; Norling et al., 2005). In a batch
mAb production process, a low pH virus inactivation step is a robust
process that is routinely used for retrovirus inactivation (Bolton,
Selvitelli, Iliescu, & Cecchini, 2015) for proteins, which are stable at a
transient low pH. However, in a continuous chromatography process
where the ProA eluate is collected continuously from different
columns at different times, there is a risk of the eluate being held for
a longer or shorter duration at low pH. mAbs are prone to
aggregation when held at low pH for extended periods (Mazzer,
Perraud, Halley, O’Hara, & Bracewell, 2015). In an attempt to
develop a continuous viral inactivation step, Klutz, Lobedann,
Bramsiepe, and Schembecker (2016) evaluated a logarithmic reduc-
tion value approach and minimum residence time approach by using a
coiled flow inverter reactor. The study indicated that the minimum
residence time approach was more advantageous from a regulatory
perspective because ProA eluate leaves the reactor only after being
held for the required inactivation time. In late 2017, Pall Corporation
launched a fully automatic Cadence™ Virus Inactivation System (Pall
Corporation, Port Washington, New York) with the ability to perform
continuous low pH virus inactivation. This system can be pro-
grammed to continuously collect ProA eluate, titrate the eluate to
low pH, hold the eluate at low pH for a predefined time, and finally
titrate the treated eluate to high pH before discharge (Levison,
Schofield, Gantier, & Gjoka, 2017). Implementing this latest technol-
ogy in mAb production process will require further studies on
process robustness, ability to integrate with different continuous
chromatography systems and process scale‐up.
Solvent/detergent treatment and ultraviolet irradiation methods
are other alternative solutions for developing a continuous viral
inactivation step that is complementary to continuous chromato-
graphy. In a recent review of implementing alternative methods for
assuring viral safety in a continuous downstream process, Johnson
et al. (2017) have highlighted the importance of evaluating virus
clearance in relation to resin age, particularly when the chromato-
graphy resins are utilized to complete saturation during continuous

SOMASUNDARAM ET AL.
downstream processing operations. In an end‐to‐end continuous
process with sample flowing from one unit operation to another,
spiking and sampling to titer virus input for the virus clearance
validation study can be challenging. To address this challenge,
Johnson et al. (2017) proposed a four‐valve system to ensure proper
loading and mixing of viral load into viral clearance/inactivation unit
operations and periodic sampling for qPCR to quantify genome copy
numbers and virus particle levels. The virus filtration unit operation,
the last purification step before the final processing, should be
carried out in a virus negative suite with facility controls that apply to
a batch process. However, integrating viral filtration operation
carried out in an isolated suite with other unit operations for an
end‐to‐end continuous process will require additional considerations.
For example, the virus filter should be sized appropriately to handle
the higher load challenge in a continuous process or strategies on
using multiple virus filters in parallel can be developed by switching
the feed flow to the subsequent filter when flux decline is observed
in the first filter (Zydney, 2016).
5.3 | Continuous intermediate and polishing
purification
In the intermediate and polishing steps, most negatively charged
process related impurities such as DNA, some host cell proteins and
endotoxin (if present) bind to an anion exchange column, when
operating in product flow through mode (Knudsen, Fahrner, Xu,
Norling, & Blank, 2001; Li et al., 2010). The feed material for these
unit operations are often adjusted to the appropriate pH and
conductivity before loading (A. A. Shukla, Wolfe, Mostafa, & Norman,
2017). During these adjustments mAbs can undergo chemical
degradation, aggregation, and modifications through oxidation,
reduction, deamidation, isomerization, and fragmentation, resulting
in a potential change to the amount of charge and size variants
(Khawli et al., 2010; Li et al., 2010; Weitzhandler et al., 1998).
Designing a straight through chromatography step by connecting two
polishing flow through steps, post ProA, can be considered as a
potential solution to reduce the intermediate hold steps and the need
for pH and conductivity adjustments. Klutz et al. (2015) demon-
strated a straight through purification of neutralized ProA eluate
using a mixed mode and anion exchange chromatography in
flowthrough mode using a BioSMB system, with continuous sample
adjustment to reduce the ionic strength between the two‐chromato-
graphy steps. Steinebach et al. (2017) demonstrated improved
process performance with semicontinuous polishing step using a
multicolumn countercurrent solvent gradient purification process. As
an alternative to flowthrough intermediate and polishing steps,
Shamashkin, Godavarti, Iskra, and Coffman (2013) combined the
intermediate and polishing steps into a weak partition anion
exchange chromatography (WP‐AEX) and designed a semicontinuous
tandem process using ProA, WP‐AEX, and viral filtration. The precise
mobile phase requirements of WP‐AEX was achieved by high
throughput optimization of ProA elution and neutralization buffers
to achieve a stable ProA eluate pH. This prevented excessive binding
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| 2901
of product to the AEX column and to improve separation of mAbs
from high molecular weight species.
In flowthrough mode purification, compared to the resin‐based
chromatography, membranes and monolith‐based chromatography is
gaining more attention due to the use of faster flow rates due to
convective flow, without compromising on adsorption capacity. For
example, Hughson, Cruz, Carvalho, and Castilho (2017) have
established the use of six different chromatography techniques,
combining chromatography resins, membrane adsorbers, and mono-
liths for establishing a three‐step straight through purification
strategy for purifying biopharmaceutical glycoproteins with minimal
or no sample adjustments in between unit operations. Although, this
might be suitable for a lab scale process, it is critical to have sample
adjustment steps and appropriate controls to measure product pH,
conductivity and concentration at the end of each unit operation at
the manufacturing scale. The most commonly used membrane
adsorbers for intermediate and polishing steps are anion exchange‐
based membranes with positively charged quaternary ammonium
functional groups for adsorption of impurities (Zydney, 2016). The
newest generation of anion exchange membrane adsorbers contain-
ing covalently attached salt tolerant primary amine ion exchange
ligands offer more evenly distributed binding capacity with enhanced
pore accessibility and ligand density, across a conductivity range of
0–20 mS/cm. The ready scalability of membrane adsorbers in SUT
format, eliminate the need for cleaning‐in‐place steps, making them
highly complementary to continuous ProA purification in increasing
productivity.
5.4 | Continuous ultrafiltration/diafiltration
The final concentration and diafiltration of biologics DS is
regularly performed using TFF as a batch process. The biopharma
industry is targeting for high‐concentration protein formulations
(up to 120 g/L) for subcutaneous administrations (Steele & Arias,
2014). For achieving such high concentrations, the process
solution is recirculated in the system several times, causing
additional shear and stress on the protein of interest. Single‐pass
TFF (SP‐TFF) comes as an attractive alternative to traditional TFF
system by using a unique flow path design and eliminating the
recirculation loop (Ayturk & Marshall, 2016). Recently, Rucker‐
Pezzini et al. (2018) addressed a major gap in the literature by
demonstrating continuous diafiltration by integrating SP‐TFF into
a fully continuous pilot scale production train to process 1 kg mAb
in 4 days. The study reported three identical stages of diafiltration
in series to achieve 99.75% buffer exchange at a feed concentra-
tion of 15 g/L and final product concentration of 148.5 g/L. The
current limitations on the availability of different size SP‐TFF
membrane is a bottleneck in using this technology for clinical and
commercial scale process (Rucker‐Pezzini et al., 2018). The impact
of product concentration, formulation buffer type, pH, conductiv-
ity, excipient level, and process temperature on formulation offset
required to counter Donnan effect in SP‐TFF is another important
knowledge gap in the literature.
 10970290, 2018, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/bit.26812 by CAPES, Wiley Online Library on [15/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
2902 | SOMASUNDARAM ET AL.

6 | MANU FAC TURING C HAL LEN GES column with 0.5‐mm diameter to conduct lab scale experiments to
derive mass balance, scale‐up and scheduling equations. In a recent
6.1 | Valve complexity study, Gjoka et al. (2017) showed the economic advantage of
continuous processing by using a 5‐cm ProA column at a flow velocity
Equipment manufacturers have incorporated additional valves, pH and
of 360 cm/hr. The column aspect ratios (diameter‐to‐bed height
conductivity probes, UV monitors, pressure gauges and control
ratio) operated in these laboratory scale studies usually allow good
hardware in a continuous chromatography system to ensure consis-
flow distribution unlike the process scale columns (Soriano, Titch-
tent product quality. In a GMP environment, these additional
ener‐Hooker, & Shamlou, 1997). Chromatography column scale‐up
components translate to increased complexity in regular operation
typically involves increasing the diameter of the column to process a
and process automation compared to a batch process (Jagschies, 2018;
higher quantity of load at a constant bed height and flow velocity
Stock et al., 2014). This complexity increases with the increase in
(Carta & Jungbauer, 2010). The diameters of manufacturing scale
number of columns. For example, in the Cadence™ BioSMB system,
columns can go above 1 m with corresponding aspect ratios >5
consists of 240 diaphragm valves in an integrated single use product‐
(Gerontas, Lan, Micheletti, & Titchener‐Hooker, 2015). Such changes
contact flow path used from 1 to 16 columns in a process development
in the column aspect ratio increases media compression and
scale or from one to eight columns in a manufacturing scale (Bisschops,
destabilizes the bed due to the loss of wall support and pressure
Frick, & Levison, 2016). Additional column positions can be added
drop (Stickel & Fotopoulos, 2001). This diminishing wall support
using this single‐use valve cassette containing all necessary inter-
directly impacts the chromatography column packing by lowering
connections during process development. However, such flexibility
critical flow velocity. In their paper, Kaltenbrunner, Diaz, Hu, and
may not be desired in a manufacturing set‐up as it can trigger a range
Shearer (2016) have characterized column dimensions to confirm
of documentation, such as additional calibration records, sanitization
that the continuous operation with shorter bed heights allows for an
records, qualification records, deviations, and corrective and preven-
increase in productivity and a decrease in buffer consumption at
tive action plans. On the contrary, GE Healthcare’s periodic counter
manufacturing scale. However, from the review of the literature, it
current technology with a minimum of three column configuration and
was identified that the favorable aspect ratios achieved in continuous
ChromaCon’s Eco Twin GMP system with two column configuration
chromatography has not been brought to light. In this review,
promises increase in productivity with minimum valve complications
chromatography column aspect ratios were calculated (Table 4) from
(Bisschops & Brower, 2013). From a manufacturing point of view, it
different column geometries for batch and continuous process
is important to design a process with equipment that offers a
reported by Bisschops et al. (2016). The calculated aspect ratio of
balance between maximizing productivity and operational simplicity.
continuous manufacturing process is 28–50% lesser, indicating that
Moreover, the incorporation of single use sensors for pH, conductivity,
the impact on critical flow velocity in a continuous process is 28–50%
product concentration, and pressure into a single use manifolds
lesser than the batch process. This analysis of aspect ratio should
can make the implementation of these complex systems more
install more confidence in biomanufacturers for continuous process
agreeable.
scale‐up.

6.2 | Column aspect ratio

6.3 | Column integrity testing
During continuous chromatography process development, shorter
columns are used at higher flow rates to maximize productivity. In a The column integrity testing before use, during processing, and after
proof of concept study, Pollock et al. (2013) used a 5‐cm bed height several runs is essential for the assurance of the quality performance

T A B L E 4 Comparison of column geometry of batch and continuous proA chromatography

Continuous clinical Continuous commercial Continuous commercial

Parameters Batch process manufacturing manufacturing manufacturing with high titer

Column diameter (cm) 80 20 30 30

Bed height (cm) 20 7 13 15
Aspect ratio 4.0 2.9 2.3 2.0
Reduction in aspect Not applicable 28 43 50
Ratio (%)
Resin volume (L) 100 17 39 50
Specific productivity (g/ 8.17 50 25 39
L/hr)
Column diameter
Aspect ratio = Bed height
(Aspect ratio of batch process − Aspect ratio of continuous process)
Reduction in aspect ratio = (Aspect ratio of batch process)
×100

SOMASUNDARAM ET AL.
F I G U R E 4 An example of start–peak–end–width (S–P–E–W)
analysis. In this example, the conductivity gradient starts at 10 CV
and the elution peak collection beings at 20 CV. The peak start is
calculated as 10 CV from the start of the conductivity gradient to
start of peak collection. Similarly, peak apex is calculated as 20 CV
from the start of the conductivity gradient to the elution peak apex
(30 CV). Peak end is calculated as 30 CV from the start of the
conductivity gradient to the end of peak collection (40 CV). Peak
width was calculated as 10 CV, the volume between 50% peak apex
on leading edge (25 CV) and 50% peak apex on tailing edge (35 CV)
of the chromatographic system. Moreover, the regulators expect the
column to be suitably qualified before each use to ensure robust and
reproducible column performance (A. Rathore, Kennedy, Donnell,
Bemberis, & Kaltenbrunner, 2003). The measure of height equivalent
to theoretical plates (HETP) and the asymmetry (AS) is a conven-
tional method used to qualify the chromatography column (Endo,
Nagamune, Katoh, & Yonemoto, 2000; Scharl et al., 2016). In
continuous chromatography, incorporating column efficiency tests
(HETP and AS measurement) after every cycle can increase the
process time and affect the scheduling of loading and nonloading
phases. As an alternative to this efficiency test, we propose to include
the start–peak–end–width analysis, referred to as S–P–E–W, on the
process chromatograms as a measure of column integrity. S–P–E–W
is a simple quantitative characterization of elution peak size and
shape that is used to monitor variations in separation within a single
column over a period of time and across multiple columns operating
in continuous mode. As shown in Figure 4, the peak start is calculated
as the volume (in column volumes) from the start of the conductivity
gradient to the start of peak collection on the leading edge of the
peak. Similarly, peak apex and peak end values are calculated as the
volume from the start of the conductivity gradient to the elution
peak apex and end of peak collection on the tailing edge respectively.
In a continuous process, the S, P, and E measurements are useful data
to track retention shifts across different columns. A loss in ProA resin
capacity is expected with the increase in the number of cycles (Jiang
et al., 2009). In a continuous process, this loss in capacity occurs
earlier than the batch process due to overloading of the column
(Kaltenbrunner et al., 2016). Such degradation to stationary phase
resulting in retention shifts in a chromatography process (Parastar,
2015) is quantitatively identified using S, P, and E values. The final
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| 2903
parameter W, the peak width at 50% peak height, is the most
commonly used measurement to characterize chromatograms
(Hinshaw, 2013). Peak width analysis has previously been used for
rapid protein kinetic characterization (Wang, Lewus, & Rathore,
2006), to characterize the separation of polar analytes and
chlorophenols on reversed phase liquid chromatography (Y. Yang,
Lamm, He, & Kondo, 2002) and analysis of ultraperformance liquid
chromatography results (Plumb et al., 2004). The 50% peak height
value is obtained by dividing the maximum UV absorbance recorded
at the peak apex by two. The width is determined by measuring the
volume between the leading and tailing edge at the calculated 50%
height. Variations in the calculated peak width is related to variations
in the number of theoretical plates (N) and HETP values using
equations described in Spangler and Collins (1975). A narrow peak
width indicates an increase in N, while a wider peak is a result of a
decrease in N. From a regulatory perspective, biomanufacturers are
expected to demonstrate consistency in column integrity (Siu, Chia,
Mok, & Pattnaik, 2014). Satisfying this regulatory expectation, the
quantitative results obtained from the S–P–E–W analysis is a
powerful tool to measure consistency in process chromatograms
and column integrity across different columns used in a continuous
process, without increasing the process time.
The use of large‐scale prepacked columns for clinical and
commercial manufacturing is becoming a growing trend in the
biologics industry. The prepacked columns offer an economic
advantage in reducing column packing resources and associated
consumables. This advantage is more relevant for continuous
chromatography where multiple columns have to be packed. In a
recent study conducted by Grier and Yakabu (2016), a prepacked
ProA column (20 cm diameter and 10 cm bed height, axially packed)
was compared to an axially packed stainless steel ProA column with
same dimensions. In‐process performance data, including product
yield and impurity clearance was comparable between the two
columns. However, the study also reported discrepancies in the
vendor and onsite column qualification data for the prepacked
column. The difference in the results were attributed to continuous
media settling during and after shipment or system differences such
as hold‐up volume or dissimilarities in the software used for analysis.
Hence, developing a robust qualification process for using prepacked
columns will be advantageous for implementing continuous process
at manufacturing scale.
6.4 | Process characterization
The regulatory agencies strongly support continuous manufacturing,
provided the manufactures develop a well understood robust process
that can meet the product quality attributes under controlled
manufacturing steps (Nasr et al., 2017). However, the lack of
experience in getting regulatory approval for continuous bioproces-
sing creates an uncertainty in the industry (Stock et al., 2014). To
meet the regulatory levels of product quality assurance, the
continuous manufacturing process needs to be developed and
optimized under a set of critical process parameters (CPPs) enabling

2904 | SOMASUNDARAM
a rich knowledge‐based filing. In a traditional batch process, scale‐
down model and characterization studies are conducted individually
for each unit operation. However, while operating in a continuous
mode, a design space incorporating multiple unit operations is
required for an increased operational flexibility. Such a multi-
dimensional risk analysis will assist biomanufacturers to achieve a
holistic view of the process and identify CPPs interacting across all
unit operations, thereby defining a design space and control strategy
across multiple process steps. To study such an extensive design
space, biomanufacturers should consider establishing a scale‐down
model of the entire process train, rather than scaling down individual
unit operations separately. Incorporating virus clearance studies into
such extensive scale‐down model to validate the clearance across
each unit operation is another challenge for biomanufacturers.
6.5 | Process economics
Continuous processing offers the opportunity to increase manufac-
turing flexibility, potentially improve product quality by operating in
steady‐state, and reduce cost (Zydney, 2015). The allure of cost
reduction has sparked numerous analyses to determine when and
where continuous processing can be most beneficial. A comparison
study on the impact of batch versus continuous processing on COGs
for an annual production of 200 kg of mAb showed a reduction in the
downstream processing COGs by ~8€/g of mAb while the medium
requirements for continuous upstream processing increased the
upstream COGs by ~33€/g of mAb (Klutz, Holtmann, Lobedann, &
Schembecker, 2016). Therefore, a hybrid approach was recom-
mended. The advantages of a batch or a continuous or a hybrid
process in terms of direct cost (e.g., raw materials, labor, QCQA
batch release), indirect cost (e.g., depreciation, facility‐dependent
overhead), manufacturing scale (i.e., clinical/commercial phase) may
vary based on the size of the company. For example, by using a
multiattribute decision‐making approach, Pollock, Coffman, Ho, and
Farid (2017) have reported that a fully continuous and hybrid
process can be more economically favorable in early phase clinical
production for small and medium sized companies. However,
continuous processing, particularly in upstream, was not as favorable
for late phase clinical production and larger companies. For all
company sizes, a complete batch process was the most beneficial
during commercial manufacturing. Taking a slightly different
approach, Walther et al. (2015) assessed the economic benefits of
an integrated continuous biomanufacturing platform for a hypothe-
tical launch portfolio of mAbs and non‐mAbs using BioSolve process
models. The study reported 57% reduction in capital expenses and
operation expenses compared fed‐batch production with single‐use
technologies for the proposed product portfolio. Another considera-
tion from a contract manufacturer perspective is that the reduction
in process time for producing the target amount of protein for a
project can result in the availability of time and resources for taking
more projects per year, resulting in an economic advantage. As the
process economics results are derived from several general assump-
tions, it will be appropriate for biomanufacturers to customize the
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ET AL.
process economics studies to suit their facility and optimize the
facility design to convert their existing sites, rather than assuming a
fresh start (Jagschies, 2018).
7 | CO NCL USION
In recent years, biopharma companies have evaluated continuous
operation of various downstream unit operations at laboratory scale to
improve process efficiency while maintaining product quality. A major
challenge in integrating continuous upstream and downstream processes
is in identifying and developing a suitable continuous midstream strategy.
Capital expense, COGs and scale‐down modeling capabilities will play a
key role in developing a continuous midstream process. The favorable
column aspect ratios in continuous chromatography, the S–P–E–W
analysis detailed in this study, innovative resins and membranes with high
mass transfer and new chemistries, supported by rapid online analytics
will accelerate the transformation of this technology from bench‐top
success to a manufacturing reality. A holistic approach to process
development and characterization will increase the process knowledge,
allowing the industry to make an informed strategic decision of either
developing an end‐to‐end continuous process or a fusion process that
uses certain unit operations in continuous mode, while other unit
operations are operated in batch mode at manufacturing scale.
AC KNO WL EDG M EN T
The authors acknowledge research funding under the Australian
Research Council Industrial Transformation Training Centre Program
(Project ID: IC160100027).
ORCI D
Balaji Somasundaram http://orcid.org/0000-0002-4555-2605
Linda H. L. Lua http://orcid.org/0000-0002-9756-7083
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SU PP ORT IN G IN FOR M ATI O N
Additional supporting information may be found online in the
Supporting Information section at the end of the article.
How to cite this article: Somasundaram B, Pleitt K, Shave E,
Baker K, Lua LHL. Progression of continuous downstream
processing of monoclonal antibodies: Current trends and
challenges. Biotechnology and Bioengineering. 2018;115:
2893–2907. https://doi.org/10.1002/bit.26812