Received: 14 October 2020 Revised: 21 March 2021
DOI: 10.1002/biot.202000524
RESEARCH ARTICLE
Complete or periodic continuity in continuous manufacturing
platforms for production of monoclonal antibodies?
Nikhil Kateja Anamika Tiwari
Department of Chemical Engineering, Indian
Institute of Technology Delhi, Hauz Khas, India
Correspondence
Anurag S. Rathore, Department of Chemical
Engineering, Indian Institute of Technology,
Hauz Khas, New Delhi 110016, India.
Email: asrathore@biotechcmz.com, Website:
www.biotechcmz.com
Nikhil Kateja, Anamika Tiwari, and Garima
Thakur contributed equally to this work.
Abbreviations: mAb, monoclonal antibody; CEX, cation exchange chromatography; SEC, size exclusion chromatography; CV, column volume; CIP, cleaning in place; A280 , absorbance at 280 nm;
HCP, host cell protein
Biotechnol. J. 2021;16:2000524.
https://doi.org/10.1002/biot.202000524
Accepted: 22 March 2021
Garima Thakur Anurag S. Rathore
Abstract
Background: Monoclonal antibodies (mAbs) currently dominate the biotherapeutic
market. This has resulted in significant efforts towards the development of a contin-
uous integrated platform for the manufacturing of mAbs.
Main Methods and Major Results: In this study, a continuous mAb platform has been
developed consisting of an Acoustic Wave Separator, a Cadence BioSMB PD system,
a customized coiled flow reactor, a modular single-pass TFF kit, an in-line diafiltra-
tion module, and a continuous dead-end filtration skid. A three-step chromatographic
purification was performed in the platform consisting of Protein A capture chromatog-
raphy followed by an anion exchange membrane directly coupled to a cation exchange
chromatography. Two operational case studies have been executed on the platform,
namely complete continuous (“CC”) and periodic continuous (“PC”) modes of opera-
tion. The CC mode was designed to ensure that each unit operation had completely
continuous inflow and outflow by increasing the number of columns, filtration mod-
ules and tanks, while the PC mode operated in periodic pulses with scheduled flow and
hold steps. Both modes were designed to handle the same flow rate and titers from the
upstream bioreactor or fed-batch harvest tank, and were compared in terms of pro-
ductivity and operational complexity. Both modes offer viable options for continuous
processing of mAbs and result in achievement of target critical quality attribute pro-
files of the final drug product over 24 h of operation.
Conclusions and Implications: It was found that the CC mode was superior in terms of
specific productivity (20–50% higher) and consumable utilization (20% lower resin uti-
lization), while the PC mode was operationally simpler and had lower facility costs due
to significant reductions in the number of auxiliary equipment (pumps, columns, tanks,
and valves). The work successfully highlighted the pros and cons of both approaches,
and demonstrates that while several groups have amply shown the superiority of con-
tinuous processing over batch mode, there are intermediate variants which may be
optimal in a given situation.
KEYWORDS
continuous processing, integrated purification, monoclonal antibody
www.biotechnology-journal.com © 2021 Wiley-VCH GmbH 1 of 14
2 of 14
1 INTRODUCTION
Development of end-to-end platforms for continuous manufacturing
of monoclonal antibodies (mAbs) is currently of significant interest to
both academic and industrial groups in the biotherapeutic industry.[1]
Continuous manufacturing has been shown to offer several advantages
over batch manufacturing including reduced capital cost, enhanced
productivity, improved product consistency without batch-to-batch
variability, and the ability to handle high upstream process titers.[2–8]
The last decade has seen rapid growth in specialized technologies
designed to facilitate continuous mAb manufacturing. Innovations in
upstream processing include perfusion cell culture systems for contin-
uous production of mAbs at high titer[9] and continuous clarification
systems[10] such as continuous centrifugation,[11,12] alternating tan-
gential flow filtration,[13,14] and acoustic wave separation.[15,16] Down-
stream innovations include versatile chromatography setups with in-
built pumps, valves and sensors enabling integrated multi-column
operation, such as the Cadence BioSMB (Pall Life Sciences), Octave
SMB (Tarpon Systems), ÄKTA PCC (GE Healthcare), Contichrom CUBE
(ChromaCon), and BioSC (Novasep).[17] For continuous viral inactiva-
tion, customized flow reactors and hold tanks have been developed
by various groups for achieving continuous low pH conditions.[18–22]
For continuous formulation, innovative single-pass ultrafiltration and
diafiltration modules have been developed which use novel flow paths
to achieve high concentration factors and have multiple inlets to
achieve 99.9% buffer exchange in a single pass of the process material
through the module.[23–25]
Integrating these technologies has been shown to result in a power-
ful and highly versatile platform for continuous manufacturing of mAbs
with continuous cell culture, clarification, capture, viral inactivation,
sterile filtration, polishing, concentration, and formulation into the final
mAb drug product. A platform consisting of perfusion cell culture, alter-
nating tangential flow filtration for clarification, capture chromatogra-
phy on ÄKTA PCC, viral inactivation in a low pH hold tank, and polish-
ing chromatography on a custom-modified four-column ÄKTA PCC was
showcased as one of the first continuous trains running from cell cul-
ture to unformulated drug substance.[26] Several platforms for contin-
uous production of mAbs at pilot scale have also been demonstrated. A
plant consisting of continuous perfusion followed by clarification, con-
centration, and sterile filtration as the first half of downstream train,
and then capture, viral inactivation, polishing, sterile filtration, formu-
lation, and bioburden reduction as the second half of the downstream
train has been proposed.[27] The two parts of the downstream train
were separated by a cooled storage step and the entire platform was
built using only single-use equipment. Two Cadence BioSMB units were
used for capture and polishing chromatography, respectively, and recir-
culation loops were used to achieve continuous viral inactivation and
formulation. Another pilot-scale integrated continuous process from
perfusion cell culture to formulated drug substance using a BioSC
Multicolumn Chromatography system for capture chromatography, a
size exclusion chromatography column for continuous viral inactiva-
tion, and single-pass TFF modules for sterile filtration, anion exchange
polishing, ultrafiltration, and diafiltration has also been proposed.[7]
KATEJA ET AL.
Recently, a continuous non-Protein A mAb manufacturing train from
clarified fed-batch harvest to unformulated drug substance has been
demonstrated by integrating a precipitation-based capture step with
two polishing chromatography steps and viral inactivation using low pH
hold.[6]
Researchers have also developed continuous platforms with vari-
ous novel aspects of monitoring, modelling, and control to improve per-
formance and consistency. An integrated continuous lab-scale process
consisting of perfusion cell culture with filter-based cell retention, a
customized two-column capture setup, a viral inactivation loop with
continuous flow, and two semi-continuous polishing steps using multi-
column counter-current solvent gradient purification (MCSGP) on a
Contichrom CUBE Combined system has been proposed.[28] Internal
recycle loops were implemented with model-based control to improve
the performance of the bioreactor, maximize the yield of the cap-
ture step, and optimize the purity-yield tradeoff in the polishing steps.
Another group developed a continuous platform from perfusion cell
culture to formulated drug product and implemented at-line analytical
chromatography methods to monitor critical quality attributes by sam-
pling the process material at various points to facilitate control deci-
sions during continuous operation.[29] Researchers have also proposed
a continuous train consisting of a perfusion bioreactor, twin-column
capture step, viral inactivation in a customized hold tank, and single-
column polishing step, along with a supervisory control and data acqui-
sition system to digitally integrate unit operations along with analyzers
including biocapacitance probes and two at-line HPLCs.[30]
It is evident from the above mentioned literature that several
robust mAb platforms have been successfully demonstrated at both
lab and pilot scale. Each platform uses different combinations of the
underlying enabling technologies, namely continuous clarification,
multi-column capture and polishing chromatography, viral inactivation
in scheduled batch-mode or continuous flow mode, and modular ultra-
filtration, to achieve continuous operation. Major innovations have
been with respect to novel chromatography setups,[17] customized
flow reactors,[21,31] integrated multi-polishing steps,[32] model-based
optimization,[33] and advanced supervisory control systems[30,34]
to implement continuous processes. Each platform has successfully
shown benefits over batch processing in terms of process productivity,
facility size and economic costs. However, it is also evident from the
wide variety of approaches that there are multiple ways of achieving
continuity, even when the base unit operations are identical. Some
of the case studies referenced above integrated continuous loading
in capture chromatography with periodic viral inactivation,[26,28]
polishing,[28] and ultrafiltration[35] steps by implementing scheduled
hold steps, while others chose to have uninterrupted flow between
the unit operations and increased the number of columns and mem-
branes to achieve this.[7] For a user intending to create a continuous
platform, many questions would come to mind. Should we aim for
complete continuity in each unit operation, or is periodic continuity
acceptable or even preferred? For example, when integrating capture
chromatography with continuous clarification, it is generally accepted
KATEJA ET AL.
that the loading step should be continuous. However, should the
chromatographic method be designed so that elution is continuous,
or are periodic elutions also acceptable? Similar choices between
continuous or scheduled periodic flows arise in other unit operations
as well, including viral inactivation, polishing chromatography, and
formulation. What are the impacts of either choice on process produc-
tivity, facility fit, and operational ease? What considerations need to be
taken into account to integrate completely continuous versus periodic
flows with neighboring unit operations?
In this work, we have leveraged our lab-scale mAb manufacturing
train consisting of continuous clarification using acoustic wave sep-
aration, continuous capture, and polishing chromatography using a
Cadence BioSMB system, continuous viral inactivation using a coiled
flow inversion reactor,[36] and continuous formulation using single-
pass ultrafiltration and diafiltration modules, to explore these ques-
tions in a targeted manner. The same platform has been run in two
modes, complete continuous (“CC”) and periodic continuous (“PC”). In
CC mode, the inflows and outflows from each unit operation are com-
pletely continuous, with no scheduled hold time. This is achieved by
developing appropriate chromatography methods on the BioSMB with
sufficient columns for both continuous loading and continuous elu-
tion, and by placing surge tanks to dampen concentration gradients
between unit operations. In PC mode, the chromatography methods
are designed without the constraints of continuous loading and elu-
tion, and each unit operation is run with scheduled starts and stops in
sync with the rest of the continuous train. The harvest flowrate and
titer conditions are the same for each mode to allow for comparisons in
terms of process productivity, resin productivity, operational complex-
ity, and facility requirements including auxiliary equipment such as hold
tanks, pumps and valves for each unit operation as well as the overall
train.
2 MATERIALS AND METHODS
2.1 Materials
The mAb used in the study is an immunoglobulin G1 (IgG1) antibody
with a molecular weight of around 150 kDa and a pI of 8.1. 20 L
of clarified cell culture harvest containing the mAb at concentration
5.0 g L-1 were obtained from a major Indian biopharmaceutical manu-
facturer. Analytical grade chemicals including glycine, tris base, acetic
acid (CH3 COOH), sodium chloride (NaCl), sodium dihydrogenphos-
phate (NaH2 PO4 ), disodium hydrogen phosphate (Na2HPO4 ), sodium
hydroxide (NaOH), hydrochloric acid (HCl), and histidine were pur-
chased from Merck, India. Buffer solutions were prepared using deion-
ized water. All the solutions were filtered through 0.2 µm filters.
2.2 Methods
A continuous mAb production train was developed at lab scale. Cell
culture harvest was obtained in 10 L batches from a fed-batch biore-
3 of 14
actor and was continuously clarified with an Pall Cadence Acoustic
Wave Separator (AWS) followed by downstream processing steps of
Protein A chromatography, viral inactivation, sterile filtration, cation
exchange (CEX) chromatography, single-pass ultrafiltration, and single-
pass diafiltration. The chromatography steps were carried out on a Pall
Cadence BioSMB System. The continuous train was operated in two
modes, namely complete continuous (“CC”) and periodic continuous
(“PC”). Each mode was operated for 24 h and samples of the harvest
and final product were taken every 1 h and analyzed by offline ana-
lytical HPLC for quantification of mAb concentration, aggregate per-
centage, and charge variant distribution. Figures 1A and 1B illustrate
the process flow diagrams for the CC and PC modes respectively. The
operating time of 24 h per mode was selected as it was sufficient to
demonstrate the key scheduling differences between the two modes
for both the individual unit operations and the overall train includ-
ing surge tanks. The operating time can be extended in each case as
per campaign requirements, as steady state is attained in both modes
in well under 24 h. The CC mode was designed to ensure that there
was continuous inflow and outflow from all unit operations. Continu-
ous loading and elution were facilitated in all chromatography steps
by increasing the number of columns and sizing them appropriately,
and surge tanks were placed between the unit operations for damp-
ening of concentration gradients. The PC mode was designed as an
operationally simpler alternative to the CC mode where the number
of columns and surge tanks was minimized, and chromatography elu-
tions were passed to the next unit operation in periodic pulses. Further
explanations of the design of the CC and PC modes and comparison in
terms of process scheduling, resin productivity, membrane productiv-
ity, overall process productivity, and train complexity are given in the
Discussion section.
2.2.1 Continuous clarification
A Pall Cadence Acoustic Wave Separator (AWS) was used for continu-
ous clarification.[15] Four acoustic chambers were connected in series.
Feed flow rate from the fed batch harvest tank was set to 5 mL min-1
for both CC and PC mode operation. The acoustic power was adjusted
to facilitate >90% reduction of total starting cell density after the clar-
ification. Prior to the runs, the chambers and tubing were cleaned with
0.1 M NaOH and flushed with deionized water.
2.2.2 Continuous depth and sterile filtration
A customized filtration skid was used for continuous dead-end
filtration.[37] A three-way solenoid valve was used to connect three
identical filters in parallel. The solenoid valves are shown as dark blue
diamonds in Figures 1A and 1B. In-line pressure sensors were used
to monitor the feed pressure and trigger the valve to switch the flow
to a fresh filter whenever pressure exceeded 1 bar indicating filter
fouling, thus ensuring uninterrupted continuous flow. One such skid
was fitted with three Supradisc HP PDE2 (0.2–3.5 um, 22 cm2 ) depth
4 of 14
F I G U R E 1 Process flow diagrams showing the current platform for continuous mAb production adapted for (A) complete continuous (“CC”)
and (B) periodic continuous (“PC”) modes of operation
filters from Pall Life Sciences and placed after the AWS for continuous
depth filtration. Another such skid was fitted with three Acrodisc 4652
(0.2 µm, 5.8 cm2 ) sterile filters from Pall Life Sciences and placed after
the pH neutralization step post viral inactivation for continuous sterile
filtration.
2.2.3 Continuous chromatography
For continuous Protein A chromatography, MabSelectSure resin (GE
Healthcare) was used. The columns were equilibrated with 20 mM
phosphate and 150 mM NaCl buffer at pH 7.4 and then loaded with
filtered harvest. The unbound material was washed with equilibration
buffer. Product was eluted using 100 mM glycine buffer at pH 3. Six
columns of 10.0 mL each were used in CC mode, while three columns
of 24 mL each were used in PC mode. The loading conditions of clar-
ified cell culture harvest from the AWS (flow rate 5.0 mL min-1 , con-
centration 5.0 g L-1 ) were kept constant in both modes of operation to
maintain comparability for productivity analyses.
KATEJA ET AL.
For continuous CEX chromatography, Poros HS resin (Applied
Biosystems) was used with a salt gradient with pH 7, 20 mM sodium
phosphate buffer as equilibration buffer and pH 7, 20 mM sodium phos-
phate buffer containing 200 mM NaCl as elution buffer. Three columns
of 22.0 mL each were used in CC mode, while three columns of 27.0 mL
each were used in PC mode. Table S1 gives the details of the chro-
matography methods for both modes of operation, including column
specifications and duration, residence time and flow rate of the loading,
second-pass loading, wash, elution, cleaning, and equilibration steps.
The column sizing and scheduling decisions are explained further in the
Discussion section. An anion exchange (“AEX”) membrane (Sartobind
Q nano 1 mL, 4 mm bed height) was placed in-line prior to the CEX
columns such that the loading onto the CEX was performed through
the AEX membrane.
All chromatography steps were performed on a single Pall Cadence
BioSMB system using a customized Python-based control software.[38]
The software layer controlled the flow rates of all seven pumps and
the positions of all 240 valves in the BioSMB system in real time based
on chromatography scheduling methods coded into a Python-based
KATEJA ET AL.
F I G U R E 2 Schematic showing BioSMB setup for (A) CC and (B) PC mode of operation and the ultrafiltration and diafiltration configuration
along with pumps and valves for (C) CC and (D) PC mode respectively
scheduler. Any desired method or combination of methods could be
coded into the Python scheduler and executed by the system, limited
only by the capabilities of the BioSMB system, namely the physical
limitations of not more than 16 columns and not more than 7
feed/buffer streams. Real-time data collected from other on-line
or at-line process sensors could also be integrated with the control
layer to facilitate the implementation of advanced control logic such
as loading and pooling decisions based on analytical tools like NIR
spectroscopy[38] or at-line HPLC,[39] as we have demonstrated in
previous work.
Figures 2A and 2B illustrate the operation of the BioSMB by the
Python control layer for the CC and PC modes respectively. The
present case study is the first example in the literature of a single
BioSMB being used to carry out both Protein A and CEX chromatogra-
phy. The system was able to accommodate both chromatography steps
by working within the limitations of pumps and valves as follows. The
eight inlets of the BioSMB are used to supply Protein A load, Protein
A second-pass, CEX load, Protein A elution buffer, CEX elution buffer,
Protein A wash buffer, CEX wash buffer, and cleaning buffer (same for
Protein A and CEX) for both modes of operation. Both modes use an
external gradient pump for providing the CEX elution gradient, and the
Python controller is used to sync the operation of the gradient pump
with the CEX elution schedule. Both modes use five of the six outlets
of the BioSMB for Protein A elute, CEX elution pool, Protein A second-
pass, Protein A waste, and CEX waste respectively. The key differences
between the two modes are the number of BioSMB pumps and valves
used.
It can be seen from Figure 2A that the CC mode uses six of the
seven in-built BioSMB pumps, while the PC mode uses only four. This is
5 of 14
because of the extra surge tanks before and after the chromatography
operations in CC mode, requiring pumping action to both supply and
withdraw material from the surge tanks. Conversely, the PC mode does
not have surge tanks between the chromatography operations, and so
the AWS feed pump provides the driving force for Protein A loading,
and the Protein A elution pump provides the driving force for CEX load-
ing.
The number of valves also differs due to the different number of
columns. The nature of the valve manifold with 240 valves and 16 col-
umn positions makes the BioSMB a versatile device in which we found
it possible to carry out both Protein A and CEX chromatography. How-
ever, it is also possible to use two separate BioSMBs for each unit oper-
ation, as is conventional.[27,40] We have chosen to showcase the use
of a single BioSMB as it is an expensive equipment and it is of eco-
nomic benefit as well as operational advantage to limit the number of
such equipment in the continuous platform. Both PC and CC modes can
be run with either one or two BioSMBs, based on the user’s individual
preference. The design strategy and comparison of equipment, instru-
mentation and scheduling between the CC and PC modes is explored
further in the Discussion section.
2.2.4 Continuous viral inactivation
Continuous viral inactivation was achieved using low pH hold in a coiled
flow inversion reactor (CFIR) developed in-house.[36,41,42,43] The setup
consisted of tubing with internal diameter 6.4 mm (Masterflex L/S 17
tubing) wound around a central coiled axis. The length of the CFIR was
adjusted to allow a total low pH hold time of 1 h at the process flow rate
6 of 14
for both CC and PC mode operations. The pH of the Protein A elute was
already 3.7, sufficient for viral inactivation. Therefore, pH adjustment
at the entrance to the CFIR was not required. In CC mode operation,
the pH was adjusted in the surge tank at the exit of the CFIR (Tank 3 in
Figure 1A) using a peristaltic pump to deliver a constant stream of neu-
tralization solution to the surge tank at a pre-determined flow rate to
bring the pH up to 7. In PC mode operation, the flow through the CFIR
was driven by the Protein A elution pump of the BioSMB, as shown in
the scheduling chart in Figure 2B. The pH was adjusted in-line using a
Y-connector and a peristaltic pump which was triggered whenever the
BioSMB Protein A elution pump was triggered. 2 M Tris base was used
as the pH neutralization solution in both modes of operation.
2.2.5 Continuous ultrafiltration and diafiltration
Single pass tangential flow filtration (SPTFF) was used for continuous
ultrafiltration. A Cadence Single-Pass TFF Modular Kit (Pall Corpora-
tion, USA) was used. The modular setup was fitted with membranes of
regenerated cellulose with a molecular weight cut-off of 30 kDa. Each
membrane had an area of 93 cm2 . Process development was carried out
to construct the modular setups yielding the desired volumetric con-
centration factors (VCFs) for the input flowrates and concentrations in
both the CC and PC setups. The input conditions to the SPTFF mod-
ule under CC mode operation were flow rate of 3.6 mL min-1 and con-
centration of 6.1 g L-1 . Similarly, the input conditions under PC mode
operation were flow rate of 7.9 mL min-1 and concentration of 5.1 g L-1 .
Further details on the process design which gave rise to these particu-
lar flow rates in the two modes of operation are given in Section 3.1.3.
The target final concentration in both cases was 20 g L-1 , corresponding
to VCFs of 3.3x and 4.0x in the CC and PC cases, respectively. Trans-
membrane pressure was fixed at around 1 bar in both cases using a
clamp on the retentate line and measuring the feed and retentate pres-
sures using in-line pressure sensors during process development stud-
ies. The target VCF was achieved by building a modular setup with 3
membranes (2 parallel + 1 in series) for the CC case and 6 membranes
(3 parallel + 2 parallel + 1 in series) for the PC case. A Masterflex peri-
staltic pump was used to pump the material from the previous surge
tank through the modular SPTFF in both cases.
For diafiltration, a Pall Cadence In-Line Diafiltration (ILD) module
was used. The membranes were made of regenerated cellulose with
a molecular weight cut-off of 30 kDa and area of 0.11 m2 . The same
unit was used in both CC and PC modes. The retentate stream from the
modular SPTFF was connected to the feed port of the ILD module. A
six-headed peristaltic pump was used to pump the diafiltration buffer
(20 mM histidine, 5 mM acetate, pH 6) into the ILD. The flow rate of the
peristaltic pump was set to 7 times the retentate flowrate of the mod-
ular SPTFF, ensuring diafiltration up to 7 diavolumes in a single pass
through the ILD. Prior to the runs, both the SPTFF and ILD were flushed
with deionized water and normalized water permeability test was con-
ducted. Figures 3A and 3B illustrate the operation of the ultrafiltration
and diafiltration steps for the CC and PC modes, respectively. Table
S2 lists the operating parameters for the CC and PC modes, respec-
KATEJA ET AL.
tively. Further comparison of the strategy design, process scheduling,
and equipment requirements of the two modes is presented in the Dis-
cussion section.
2.3 Analysis methods
2.3.1 Analytical size exclusion chromatography
Superdex 200 column (1.0 mm × 300 mm) from Cytiva Lifesciences
was employed for size exclusion high performance liquid chromatogra-
phy (SEC-HPLC) for determination of % high molecular weight impu-
rities (HMWI%). Analysis was performed at 0.5 mL min-1 at 25◦ C
using 50 mM phosphate buffer of 300 mM NaCl, pH 6.8 as mobile
phase for a runtime of 45 min. Samples, before injection, were filtered
using 0.2 µm, syringe filter. 10 µg was injected for each sample. UV
absorbance at 280 nm was monitored.
2.3.2 Analytical affinity chromatography
Analytical protein A affinity chromatography was performed for mAb
quantification. A Bio-Monolith Protein A (4.95 × 5.2 mm) column from
Agilent Technologies was used for the analysis. Analysis was performed
at 0.5 mL min-1 at 25◦ C using equilibration buffer (50 mM sodium phos-
phate, 150 mM sodium chloride at pH 7.5) and elution buffer (50 mM
sodium phosphate, 150 mM NaCl, pH 2.5). For analysis, 5 µg of filtered
sample was injected and UV absorbance was monitored at 280 nm.
Antibody concentration was calculated using a calibration curve (peak
area vs. protein amount) prepared form pure mAb sample of known
concentration.
2.3.3 Analysis of charge variants
Levels of charge variants in the various samples were measured using
analytical cation exchange high performance liquid chromatography
(CEX-HPLC). An Agilent Bio MAb NP5 (4.6 × 250 mm, Agilent technolo-
gies, India) column was connected to Agilent 1260 liquid chromatogra-
phy system. Analysis was performed at 0.5 mL min-1 at 25◦ C using equi-
libration buffer (15 mM sodium phosphate, pH 6.2) and elution buffer
(150 mM sodium phosphate, pH 6.2). The method used for analysis of
charge variants has been published elsewhere.[44] UV absorbance at
280 nm was monitored.
2.3.4 HCP and DNA measurement
Quantification of residual DNA and host cell proteins (HCP) in the
purified mAb samples was performed using the Quant-iT PicoGreen
DNA kit (Invitrogen, UK) and CHO HCP ELISA kit (Cygnus Technolo-
gies, USA) respectively. All the reagents and buffers were prepared
according to manufacturer’s protocols. Detailed methods for both
KATEJA ET AL.
F I G U R E 3 Scheduling charts for the operation of the mAb platform in (A) CC mode and (B) PC mode including details of the chromatography
and ultrafiltration sub-steps. Text in red gives details of the time (“t”) of each step in min and the flow rate (“u”) of each step in mL/min
these analyses have been laid down in our previous publications and
the same were followed.[6,42]
3 RESULTS AND DISCUSSION
3.1 Leveraging the mAb production platform to
achieve varying levels of continuity
The aim of the present work was to use the same mAb platform con-
sisting of an Acoustic Wave Separator (AWS), a 16-column BioSMB PD
system, a coiled flow inversion reactor (CFIR), a modular single-pass
ultrafiltration (SPTFF) device, and an in-line diafiltration (ILD) module
to demonstrate two variants of continuous operation, namely complete
continuous (“CC”) and periodic continuous (“PC”), and compare the
resulting processes in terms of process productivity, consumable pro-
ductivity, operational complexity, and facility fit, under near-identical
7 of 14
conditions of feed flowrate and titer from a fed-batch harvest. The pro-
cess flow diagrams for the two modes have been presented in Figure 1
and the detailed process scheduling charts are illustrated in Figure 3.
The CC mode was designed to ensure that both inflow and out-
flow from each unit operation was completely continuous (Figure 3A).
Thus, the AWS was continuously supplied with material from the fed-
batch harvest tank at a rate of 5 mL min-1 , and the outflow was con-
tinuously loaded onto the BioSMB for Protein A capture chromatog-
raphy. The capture method was designed to ensure that at least one
column from the multi-column setup was always undergoing elution.
Full elutions were collected in a surge tank with constant flow into the
CFIR for viral inactivation. The CFIR outflow was therefore also con-
tinuous and led to a surge tank where the pH was neutralized, prior
to continuous loading onto the same BioSMB for CEX chromatography
in bind-and-elute mode (Figure 2A) via a flow-through AEX membrane.
Once again, the polishing method was designed to ensure that at least
one column in the multi-column setup was always undergoing loading
8 of 14
and another elution. Volumetric pooling was performed on the elutions
and the pools were collected in a surge tank. From this tank, the mate-
rial was continuously pumped through an SPTFF+ILD setup for for-
mulation followed by storage of the final drug product. Since the flow
through the SPTFF+ILD was required to be uninterrupted, a parallel
set of modules was required to which the process flow stream could be
switched during periodic membrane cleaning operations every 8–12 h
(Figure 2C). All the tanks in CC mode were maintained at five times the
elution volume of the preceding step to dampen concentration gradi-
ents in the elution peaks.
In the PC mode of operation, the loading onto Protein A chro-
matography was performed continuously by designing a multi-column
method where one column was always in the loading phase. This was
because continuous loading onto capture chromatography is an essen-
tial requirement of continuous platforms so that they are capable of
handling continuous upstream perfusion processes. All the subsequent
unit operations were controlled by the Protein A elutions, without any
additional constraints of continuous inflow or outflow. Therefore, the
overall process flow through the continuous platform was in a pulsed
manner, driven by the schedule of the elutions from the Protein A
columns (Figure 3B). Protein A elution was directly loaded onto the
CFIR and subsequently onto the CEX columns after in-line pH adjust-
ment without any intermediate hold tanks followed by membrane AEX
in flow through mode. Each elution was essentially a pulse of material,
with a 1:1 relationship between the Protein A and CEX columns, and
no mixing between pulses. The SPTFF+ILD setup was run as shown
in Figure 2D, with cleaning operations scheduled in the pause time
between CEX elution pulses. A tank was required between the CEX
elution pool and the SPTFF+ILD setup to prevent concentration gra-
dients in ultrafiltration feed stream. The tank was also operated in a
pulsed manner, with one CEX elution pool being completely processed
by the SPTFF+ILD modules prior to the next elution being collected in
the tank, as shown in Figure 3B.
3.1.1 Design of continuous capture
chromatography in CC and PC modes
One of the major benefits of continuous capture chromatography is
the ability to achieve higher binding capacities by placing a second-
pass column in series after the main loading column to capture break-
through material and enable loading up to 70–90% breakthrough, com-
pared to batch-mode where there is only one column and loading is
done only up to 5–10% breakthrough to prevent loss of product.[6,45,46]
This principle of periodic counter-current chromatography (“PCC”)
was applied to both CC and PC modes of operation. The loading flow
rate and titer was fixed at 5.0 mL min-1 and 5.0 g L-1 , and elution
was fixed at 5 column volumes and 2.4 min residence time in both
modes (Table S1). This resulted in an elution of 12 min duration in both
modes. In CC mode, the duration of the loading and elution steps were
matched in order to facilitate both continuous loading and elution. This
led to the development of a six-column method where each of the six
columns cycled through loading, wash, elution, cleaning, equilibration,
KATEJA ET AL.
and second-pass loading in sequence, with each step of 12 min length
(Figure 3A). Given these constraints of loading time, flow rate and titer,
the Protein A columns were sized at 10 mL volume (12.7 cm height,
1 cm diameter) in CC mode, resulting in a loading residence time of 2
min and resin binding capacity of 30 g L-1 .
The PC mode was designed to minimize operational complexity.
One of the simplest modes of continuous Protein A chromatography
is the three-column periodic counter-current (“PCC”) method.[45] In
this mode, the loading step determines the cycling rate of the columns.
The durations of the non-loading steps (wash, elution, cleaning, and
equilibration) are set so that their combined duration is equivalent
to that of the loading step. This ensures a cyclical operation where
one column is always in the loading phase and another is in second-
pass while the third undergoes all the remaining steps. Matching the
elution, wash, cleaning, and equilibration steps with those in the CC
mode resulting in a loading time of 48 min, and columns were sized
at 24 mL volume (12 cm height, 1.6 cm diameter) to match the height
as closely as possible with the columns in the CC mode. This resulted
in a loading residence time of 4.8 min and resin binding capacity of
50 g L-1 . In both PC and CC mode, Protein A columns were oper-
ated at 70–80% breakthrough, as is optimal for continuous Protein A
chromatography.[6,45,46] It is evident that there was a large difference
in Protein A resin binding capacity between the CC mode (30 g L-1 ) and
the PC mode (50 g L-1 ). This is because both trains were designed to
handle the same product flow from upstream in g/min, so as to maintain
comparability of the overall train by having the same g per min for the
entire process. Therefore, the necessity of having continuous elution
to fulfil the requirements of complete continuity led to faster cycling
times in Protein A, imposed a constraint on the system to have smaller
columns and therefore less residence time in CC mode, impacting the
binding capacity. In PC mode, the loading step was much longer as it
was no longer required to have continuous elution, and the residence
time was lengthened, leading to higher binding capacity.
The BioSMB setups used to implement the six-column capture step
in CC mode and three-column capture step in PC mode are shown in
Figure 2, and the scheduling for both modes are detailed in Figure 3. It
should be highlighted that the fixed constraints of load flow rate, load
titer, elution column volumes, and elution residence time result in sig-
nificantly different capture step design in both modes. Adding the addi-
tional constraint of continuous elutions to the CC mode led to doubling
of the number of columns, and the necessity of matching the durations
of the loading and elution steps.[6] This resulted in a shorter loading
step with reduced residence time and binding capacity. The average
concentration of the elute in the CC mode was 5.7 g L-1 , significantly
lower than the value of 9.5 g L-1 in the PC mode. However, the total
resin utilized was lower in the CC mode by around 17%.
3.1.2 Design of continuous polishing
chromatography in CC and PC modes
The flow rate and concentration of the CEX load was determined by
those of the elution of the capture step, which had flow rates of 4.2 and
KATEJA ET AL.
10.0 mL min-1 , and average concentrations of 5.7 and 9.5 g L-1 in the
CC and PC modes, respectively. The elution conditions were fixed at 15
column volumes and 3 min residence time in both modes, with pooling
of 7.5 column volumes. This resulted in an elution time of 45 min for
both modes. Once again, the constraints of continuous loading and elu-
tion in CC mode led to significant differences in the polishing method
compared to that of the PC mode. Three columns with equal duration
of loading and elution steps were sufficient to achieve the desired con-
tinuity in both steps, as shown in Figure 3A. Under the constraints of
45 min loading time, 4.3 mL min-1 load flowrate and 5.7 g L-1 load con-
centration, the CEX columns in CC mode were sized at 21.6 mL volume
(10.8 cm height, 1.6 cm diameter) with binding capacity of 51 g L-1 and
a loading residence time of 5 min.
For PC mode, the CEX method was designed so that the elution from
the first capture column would be completely loaded on to the first CEX
column, and so on for each of the three columns. The CEX loadings were
therefore in the form of periodic pulses synced to the Protein A elu-
tions. Since the duration of the Protein A elution in PC mode was 12
min, this was the required loading time. Under the constraints of 12 min
loading time, 10.2 mL min-1 load flow rate and 9.5 g L-1 load concentra-
tion, the CEX columns in PC mode were sized at 27 mL volume (13.3 cm
height, 1.6 cm diameter) with binding capacity of 43 g L-1 and loading
residence time of 2.7 min. The elution, wash, cleaning and equilibration
conditions were the same as those in PC mode.
The BioSMB setups used to implement the three-column setups in
CC and PC modes have been shown in Figure 2, and the scheduling for
both modes are detailed in Figure 3. The sizing and number of columns
was determined by the capture step in both cases, though the addi-
tional constraint of continuous elution in CC mode led to some process
design differences. The average concentration of the elute in the CC
mode was 6.1 g L-1 , slightly higher than the value of 5.1 g L-1 in the
PC mode. The total resin utilized was lower in the CC mode by around
20%. Also, there was a non-productive time of around 42 min for each
column in PC mode, as shown by the white space in the scheduling
chart in Figure 3B. This was due to the necessity of waiting for Pro-
tein A elutions in order to load the CEX columns due to the pulsed
approach.
3.1.3 Design of continuous ultrafiltration and
diafiltration in CC and PC modes
The input conditions to the SPTFF module were determined by the
elution output of the CEX step. Under CC mode operation, the feed
flow rate to the SPTFF was 3.6 mL min-1 and concentration was 6.1 g
L-1 . Similarly, the input conditions under PC mode operation were
flow rate of 7.9 mL min-1 and concentration of 5.1 g L-1 . In single-pass
ultrafiltration, the target concentration must be achieved in a single
pass through the module. This is done by increasing the membrane
length by having several smaller membranes in various series and
parallel configurations. Moreover, the flow rate through the module
and the concentration of the input feed material critically affect the
volumetric concentration factor (VCF) achieved in a single pass, as they
9 of 14
affect the residence time and permeate flux respectively.[35] Since
both the feed flowrate and the feed concentration in the CC and PC
modes were constrained by the CEX elution, it was necessary to design
the SPTFF module configuration such that the output concentration
target of 20 g L-1 was achieved, corresponding to VCFs of 3.3x and
4.0x in the CC and PC cases, respectively. The module configurations
were scouted experimentally and it was determined that a 2 parallel
+ 1 series configuration was appropriate for the CC case, while a
3+2+1 configuration was appropriate for the PC case to achieve the
required concentration target.
Smooth operation of the ultrafiltration and diafiltration step in
the continuous train requires not only proper membrane sizing and
module configuration, but also setups for periodic buffer flush and
cleaning of the membrane modules. For the CC mode, both ultra-
filtration and diafiltration were carried out continuously with no
pause time. Therefore, a parallel SPTFF+ILD setup was required to
facilitate cleaning without disruption of the flow stream, as shown in
Figure 2C. For the PC mode, the flow through the membrane modules
was periodic. Therefore, cleaning could be scheduled during periods
of non-operation without requiring a parallel membrane setup, as
shown in Figure 2D. It can be seen that the CC mode requires a total
of six valves to enable process material to be directed through one
set of SPTFF+ILD membranes, while the other undergoes buffer flush
and cleaning. Conversely, the PC mode requires only two valves as
continuous flow is not a constraint, allowing processing, buffer flush
and cleaning to be scheduled sequentially. The CC mode also requires
an extra pump as both material processing and flush/cleaning are
carried out simultaneously. Both modes have a total of four tanks for
the process material, flush buffer, cleaning buffer, and formulation
buffer, respectively.
3.1.4 Comparing start up and shut down times in
CC and PC modes
The presence of surge tanks also impacted the start-up and shut
down times of the two modes. All the surge tanks in CC mode were
maintained at 5x the elution volume of the preceding step, except
the first tank which was maintained at 5x the loading volume of the
Protein A step. Protein A loading was started after ST01 had filled to
300 mL, taking 1 h. Then, loading onto the CFIR was started after five
Protein A elutions were collected in ST02, also taking 1 h. The CEX was
started after five CFIR outputs had collected in ST03, taking another
1 h. Finally, the ILC and ILD were started after five CEX elutions were
collected, taking an additional 4 h. Therefore, the total time for the
CC mode to reach steady state was 7 h. Conversely, the lack of surge
tanks in PC mode allowed it to reach steady state as soon as the first
cycle was completed. The first Protein A elution passed through the
CFIR and onto the CEX columns immediately, and the subsequent CEX
elution immediately passed through the ILC and ILD. The total start
up time was therefore equivalent to the time of Protein A loading,
which is just 48 min, after which there was no difference between
start-up operation and steady-state operation due to the periodic
10 of 14
scheduling. The logic for shut-down time can be similarly constructed,
and shut-down is much faster for PC mode compared to CC mode.
Though PC mode is much faster to start-up and shut-down, there
are few options for at-line process monitoring as there are no surge
tanks where the product is collected without gradients and easily
accessed by immersion probes, though in-line options for monitoring
are possible in both CC and PC modes. There is also limited scope to
pause individual unit operations and carry out repairs without stop-
ping the whole train, due to the lack of tanks for holding intermediate
material.
3.1.5 Product quality results in CC and PC modes
of operation
The continuous platform was successfully demonstrated for both CC
and PC modes over 24 h runs, with the final drug product consistently
achieving the target critical quality attribute profile of 20 g L-1 mAb
concentration, 15% acidic species content and <0.2% aggregate
content, as shown in Figure 4A which summarizes the offline analytics
carried out every hour during the continuous operation. Further,
HCP concentration of <10 ppm and DNA concentration of <10 ppb
were obtained in the pooled drug product for both CC and PC mode,
compared to the initial harvest which had HCP content of 92,811
± 4919 ppm and HCDNA content of 978 ± 64 ppm. Representative
elution chromatograms for the capture and polishing steps are shown
in Figure 4B. Finally, it may be required to have an additional step for
viral filtration at the end of the process. A similar skid as described
in Section 2.2.2 for continuous dead-end depth and sterile filtration
can be implemented for continuous viral filtration. There would be
no significant difference between the CC and PC modes as it is a
simple flow-through membrane separation with minimal flow rate
constraints. Alternatively, batch-mode viral filtration of the pooled
drug product at the end of either the CC or PC mode continuous train
could be done at the end of the continuous campaign or infrequently
throughout.
3.2 Considerations for choosing between
complete and periodic continuity
It is clear that both CC and PC are viable modes of operation for the
continuous mAb platform. Both were able to handle the same upstream
flow rate and titer and achieve the target critical quality attribute pro-
file in the final drug product. The basic methods of the unit operations
were the same in both modes, though there were differences in the col-
umn sizing, membrane sizing, number of columns, number of filtration
modules, and number of auxiliary equipment including tanks, pumps
and valves between the two processes. One of the useful metrics of
comparison between different processes is productivity—both overall
process productivity and specific productivity of different unit opera-
tions. Multiple researchers have compared these parameters between
batch and continuous processes, and continuous processes have gener-
KATEJA ET AL.
ally shown significant productivity gains of 20–50%.[6,45,46] Figure 4C
shows the results of a similar productivity comparison between the CC
and PC modes of operation of our platform. The total process produc-
tivity was calculated from the retentate stream of the ILD module. In
CC mode, the ILD retentate stream has a constant flow rate 1.1 mL
min-1 and concentration of 19.9 g L-1 , while in PC mode the flow rate
and concentration are 20.1 g /L-1 and 2.0 mL min-1 with periodic flow
(25.5 min on and 22.5 min off), as shown in the scheduling charts in Fig-
ure 3. This resulted in the same total productivity of 21.8 g mAb pro-
duced per minute in both modes. This is expected as both platforms
are operating at steady-state and are processing the same amount of
upstream material, namely 5.1 g L-1 at 5.0 mL min-1 flowrate equivalent
to 25.5 g mAb per minute incoming from the upstream fed-batch tank
with 95% yield in the capture step and 90% yield in the polishing step.
However, CC mode had higher specific productivity for Protein A
resin, CEX resin, and SPTFF membrane area. The faster cycle times and
smaller column volumes in CC mode, designed to facilitate both contin-
uous loading and continuous elution, allowed higher productivity per
unit resin volume in the chromatography steps. The specific produc-
tivity for Protein A and CEX were around 20% and 29% higher in CC
mode compared to PC mode, respectively, in units of mg elute/min/mL
resin. In the case of specific productivity of SPTFF, the continuous flow
in CC mode facilitated lower flowrates compared to the PC mode,
where the operation in the form of periodic pulses and pauses led to
higher flowrates during the periods of operation to compensate for the
periods of hold. As explained in Section 2.2.5, the differences in feed
flowrate and feed concentration necessitated different SPTFF module
configuration to achieve the same target concentration of 20 g L-1 in
both modes. This led to a total productivity difference of 52% in the CC
mode compared to PC mode in units of mg retentate/min/cm2 STPFF
membrane area.
It is clear that the complete continuous mode of operation offers
superior specific productivity due to the lower consumable require-
ments of resin and membrane area, and that both modes have the same
overall process productivity governed by the upstream material flow.
Any combination approaches, such as capture chromatography in CC
mode followed by polishing in PC mode, would result in reduction in
specific productivity, while the total productivity remains unchanged.
However, it is evident from the process flow diagrams in Figure 1 and
the scheduling charts in Figure 3 that the PC mode is operationally sim-
pler than the CC mode. These differences are summarized in Table 1. It
is evident from the facility related parameters in Table 1 that the opera-
tional simplicity of the PC mode results in economic advantages on the
facility front. Fewer vessels, columns, SPTFF modules, and pumps are
required, decreasing the capital investment cost. Another point to note
is that while the lab-scale version of the BioSMB can accommodate up
to 16 columns, the pilot-scale version of the equipment can only accom-
modate up to eight. Since the CC mode of operation has a total of nine
columns, implementing it at pilot scale would necessitate an additional
BioSMB system at a significant capital cost. This would not be required
for the PC mode which has only six columns in total.
In summary, increasing level of continuity results in increasing
specific productivity and consequentially lower cost of manufacturing.
KATEJA ET AL. 11 of 14

F I G U R E 4 (A) Critical quality attributes of mAb concentration, % acidic species and % aggregate content measured in the fed-batch harvest
and final drug product for the two modes of operation, (B) Representative elution chromatogram for the Protein A and cation exchange
chromatography in the two modes of operation along with analytical SEC and CEX-HPLC profile for the final drug product, (C) Comparison of total
and specific productivities for the two modes of operation
12 of 14 KATEJA ET AL.

TA B L E 1 Comparison of the two modes of operation in terms of 4 CONCLUSIONS

facility and consumables requirements

Facility Related Parameters A mAb platform has been developed consisting of continuous clari-

Parameter CC Mode PC Mode
No. of tanks 4 1
No. of protein A columns 6 3
No. of CEX columns 3 3
No. of SPTFF modules 2 1
No. of ILD modules 2 1
No. BioSMB pumps used (out of 7) 6 4
No. of BioSMB valves used (out of 240) 75 48
No. of auxiliary pumps 6 4
fication, depth filtration, capture chromatography, viral inactivation,
polishing chromatography, and formulation. A series of enabling
technologies have been used as the building blocks of the platform,
including an Acoustic Wave Separator, a Cadence BioSMB PD system,
a customized coiled flow reactor, a modular single-pass TFF kit, an
in-line diafiltration module, and a continuous dead-end filtration
skid. Two operational cases were executed on the platform, namely
complete continuous (“CC”) and periodic continuous (“PC”) operation.
The CC mode was designed to ensure that each unit operation had
completely continuous inflow and outflow by increasing the number

No. of auxiliary valves 8 4

Consumables
Volume of protein A resin x 1.2x
Volume of CEX resin x 1.2x
Volume of AEX membrane x x
Membrane area per SPTFF module x 2x
However, so does the complexity of hardware and operations and
as a result there may be situations where an intermediate level of
continuity may be more suitable. For example, in CC mode, the Protein
A resin undergoes a larger number of cycles compared to the PC mode.
In the current case studies, the Protein A cycle time is 12 min in CC
mode and 48 min in PC mode, resulting in a 4x faster rate of cycling.
This is a potential benefit in the case of manufacturing for clinical
trials, where a single manufacturing campaign is planned and it is desir-
able to fully exhaust consumables at the end of the campaign.[47,48]
Another potential benefit of CC mode is the large number of tanks
which provide opportunities for in-process monitoring in surge tanks
where the product is collected without in-process gradients and easily
accessible by immersion probes or conveniently accessible for at-line
HPLC analysis, in line with US FDA guidelines on Quality by Design in
biopharmaceutical manufacturing[49,50] by monitoring critical quality
attributes and critical process parameters throughout the continu-
ous train. On the other hand, the PC mode offers the advantage by
preventing mixing of process streams in any of the tanks. The pulsed
approach in PC mode allows for easy definition of a “batch” as a single
Protein A elution pulse passes through the subsequent unit operations
of viral inactivation, polishing chromatography, sterile filtration and
formulation without ever being mixed with other pulses. This 1:1
matching between capture and elution columns mitigates the effect of
column failure or in-process errors such as wrong elution gradient, and
allays many of the concerns felt by the batch-processing community
in terms of the risks associated with pooling drug substance with
different product quality profiles in continuous processes.[51] The final
decision of going for complete, periodic or hybrid continuity in a mAb
platform process should be taken after due consideration of these
factors.
of columns, filtration modules and tanks, which is what is typically
expected from the term “continuous processing”. Alternatively, the PC
mode operated in periodic pulses in line with the intrinsic periodicity of
chromatography unit operations, where loading and elution are always
sequential. Both modes are viable options for continuous processing
of mAbs and allow the target critical quality attribute profiles of the
final drug product to be achieved over 24 h of operation.
The two cases were designed to highlight the variety of approaches
in which continuity can achieved in mAb processing, even when the
underlying platform is identical. Both modes were designed to han-
dle the same flow rate and titers from the upstream bioreactor or
fed-batch harvest tank, and were compared in terms of total process
productivity, specific productivity of the unit operations in terms of
resin and membrane utilization, and operational complexity in terms
of advanced scheduling requirements and number of auxiliary equip-
ment including tanks, valves and pumps. It was found that the CC mode
was superior in terms of specific productivity and consumable utiliza-
tion, while the PC mode was operationally simpler and had lower facil-
ity costs due to significant reductions in the number of auxiliary equip-
ment. The work successfully demonstrates that while superiority of
continuous processing over batch mode is evident, varying extent of
continuity can be achieved. It is of benefit to both industrial and aca-
demic researchers to be aware of the pros and cons of different types
of continuity when designing processes on their respective continuous
platforms using similar enabling technologies.
ACKNOWLEDGMENTS
Authors would like to acknowledge funding support from Depart-
ment of Biotechnology, Ministry of Science and Technology (num-
ber BT/COE/34/SP15097/2015) and Ministry of Human Resources
and Development (Uchchatar Avishkar Yojana/MHRD 21–105/2015-
TS.II/TC). Nikhil Kateja would like to thank Council of Scientific
and Industrial Research (CSIR), New Delhi (India) for financial
assistance in the form of Research Associate Fellowship (File no.
09/086(1363)/2019-EMR-1). Anamika Tiwari and Garima Thakur
would like to acknowledge support from TCS Research Scholar Pro-
gram (RSP) cycles 14 and 15, respectively.
CONFLICT OF INTEREST
The authors declare no financial or commercial conflict of interest.
KATEJA ET AL.
AUTHOR CONTRIBUTIONS
Nikhil Kateja: conceptualization; investigation; methodology; writing-
original draft. Garima Thakur: conceptualization; investigation;
methodology; writing-original draft. Anamika Tiwari: conceptualiza-
tion; investigation; methodology; writing-original draft. Anurag S.
Rathore: Conceptualization; funding acquisition; project administra-
tion; supervision; writing-review and editing.
DATA AVAILABILITY STATEMENT
Data available on request from the authors. The data that support the
findings of this study are available from the corresponding author upon
reasonable request.
ORCID
Anurag S. Rathore https://orcid.org/0000-0002-5913-4244
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SUPPORTING INFORMATION
Additional supporting information may be found online in the Support-
ing Information section at the end of the article.
How to cite this article: Kateja, N., Tiwari, A., Thakur, G., &
Rathore, A. S. (2021). Complete or periodic continuity in
continuous manufacturing platforms for production of
monoclonal antibodies?. Biotechnol J, 16:e2000524.
https://doi.org/10.1002/biot.202000524