GENengnews.com|JUNE 18, 2019

Sartorius Takes a Single-Use Approachto Derive Value from Biomanufacturing
By K. John Morrow Jr., PhD

Some of the key challenges facing the biotech industry wereexamined recently at a Sartorius
Stedim Biotech (SSB) Conference in San
Francisco. Miriam Monge, head of themAb and recombinant protein market
segment at SSB, and chair of the conference, emphasized four critical issues:a pressing need for cost reduction; theimportance of flexibility in the design offacilities; meeting the demands of speed
to the clinic through development andtech transfer; and precise measurementof product quality in order to meet thedemands of the FDA and other agencies.
Weichang Zhou, PhD, senior vp at
WuXi Biologics, gave the keynote address and discussed his company’s development of an accelerated timeline, down
to eight months from 25, for producing
biologics in high quantities. By employing 2,000 L disposable bioreactors they
achieved productivity that was comparable to 20,000 L stainless steel bioreactors, resulting in significant reductions
in cost of goods. The platform is basedon continuous cell culture and continuous direct product capture and can bescaled up to generate clinical levels ofproduct. According to Zhou, “Scale outstrategies provide an effective solution tomeet product demands during productlife cycles and eliminate cell culture scaleup risks.”

Process intensification
Gerben Zijlstra, PhD, global techexpert at SSB, noted that “process intensification enables the use of flexible andlow-cost single-use facilities for medium
to high output commercial manufacturing.” To optimize cell output, the Sartorius Cellca Cell Line Generation team
employs ambr® systems, and has foundthem to be quite useful, saving time andresources in large scale screening andoptimization protocols. This strategy
allows selection of the optimal clone forprocess intensification at an early stage.
In addition, the use of 100 mL cryobags
as process intermediates to reduce theseed train was demonstrated successfully.
To evaluate the range of intensified
cell culture approaches, including N-1

and main bioreactor perfusion, the team
set up modeling experiments employing
ambr 15 screening workstations fittedwith centrifuge inserts. To intensify theupstream expression in the main bioreactor, high volume, high inoculation protocols from N-1 perfusion were chosen.
This auspicious choice yielded a 2-foldproductivity increase on a standard fed
batch protocol when selecting the appropriate clone (from 4 g/L in 14 days toover 6 g/L in 10 days).
The team also adopted an intensified
fed batch approach, with one volume
per day of media exchange using the centrifugal containers that are available withthe ambr system. This protocol was determined to be the most productive, witha high level of production of >13 g/L in12 days (from 3 g/L in 12 days). And
by identifying the best clones, cumulative titers of up to 20 g/L were achieved
at 1VVD medium exchange and onlyaround 30 million cells/mL.
Using the ambr 250 platform, bothinternal SSB teams as well as large biopharma customers were able to ramp
up their cell numbers to over 100 x 106
cells/mL. Also, extended cultivation
lengths of 30 days have been demonstrated and customers have been able toshow comparable product quality results
to existing small scale models (benchtopbioreactors).
“Therefore, we have found thatwhen using this new ambr perfusion
toolbox in cell line and process development, there is no longer a fundamental
roadblock for efficient developmentof intensified, perfusion-based process
platforms, in similar time frames as
current fed batch process platforms,”
stated Zijlstra.
The overall improvements in upstream productivity were achievedby use of Sartorius tool sets for theoptimization of various aspects of theintensified biomanufacturing process;the ambr development platforms, theRM and STR perfusion enabled manufacturing platforms, and simultaneous
media and process enhancements.
“With consistent effective titers of 10g/L and beyond, annual outputs ofgreater than 1,500 kg from intensified
single-use facilities can be realized,”
Zijlstra concluded.
Scalable single-use platforms“The huge gains in cell culture
productivity drive efforts to catch up
performance at the downstream end;
this is a constant, ongoing process,” said
Thomas Erdenberger, global tech consultant at SSB. “Today we find ourselves
behind the eight ball again, so at thispoint we employ the tools worked out

by our company to advance the speed
and efficiency of the downstream performance.”
The success at producing high celldensities at the upstream end means thatthe biomass must be removed withoutliberating host cell proteins into the culture medium. Erdenberger described thekSEP platform, a fully single-use, closed
system with gamma irradiated consumables for all product contact surfaces. It
is capable of clarifying high cell density
mammalian cell cultures with low shear,minimizing cell lysis and release of hostcell proteins. It is fully automated andcan perform as a continuous operation
during the primary recovery step. In
model experiments, Erdenberger and hiscolleagues demonstrated cell high density clarification (108 cells/mL) with fullproduct recovery (>95%) and no significant viability loss or cell lysis.
Erdenberger discussed the issue ofviral clearance and advances made inthis area, including the application ofa simplified virus filtration unit with asingle-use transfer assembly. This unit ispre-assembled, pre-sterilized, ready-touse (so called “plug-and-play”) and isdesigned for application in high intensity
transfer situations.
The use of flow-through polishing for
impurity removal through a two-steppurification process of mAbs was also
covered in Erdenberger’s presentation.
A high level of removal of aggregates
was achieved using Sartobind S cationexchange (CEX) in a high flow-throughmode. In model experiments with
Murine leukemia virus there was very
good LRV and removal of impurities by
AEX and salt-tolerant AEX membraneadsorbers, including host cell proteins(HCPs), and DNA.
Carrying forward the discussion ofcontinuous bioprocessing, Anna Persson, principle data scientist at SSB,
emphasized that “continuous and2 | JUNE 18, 2019 | Genetic Engineering & Biotechnology News | GENengnews.com

intensified bioprocessing is attracting
much attention from biopharmaceuticalcompanies seeking to maximize the efficiency of their manufacturing assets.”
Benefits of this approach include smaller
facilities, reduced scale up risk, moreconsistent product quality, and higher
throughputs.
To drive the data analytical process
forward, Persson takes advantage ofUmetrics Suite of Data Analytics Solutions, a common, large-scale, automatedplatform designed for handling large
quantities of data. Through the mining
and modeling process, predictive poweris generated, making possible large
improvements in real life situations. A
challenge put forward by Lonza wasa record of lower than expected average yield in their facility, which madeit difficult to plan work and delivery
to the end customers. Employing variability analysis allowing optimization ofyield and time-based multivariate data
analysis of real-time process parameters
increased yield and decreased the cycle
time, resulting in a significant increase ofplant output.
Similar strategies were put to use in aproject with Amgen, in which viable celldensity was improved 23% and anothereffort with Novartis, analyzing Out ofControl Incidents, allowing improvement of process consistency.
According to Persson, multivariate
analysis is essential in value generation,and regulatory agencies are now encouraging use of tools such as the Umetrics
Suite. “Looking to the future, we foresee the use of mechanistic modeling anddeep learning, and an expansion into
hybrid modeling,” Persson stated.
Moving manufacturing productivity
beyond the initial facilities’ capabilities
requires a fundamental understanding of the needs of large bioprocessing
operations, explained Priyanka Gupta,
bioprocessing modeling manager at

SSB. Gupta directs a team that analyzes
production processes in order to define
the most cost-effective framework.
They employ Biosolve software as amodeling tool to select the most significant impact on overall process economics. The team examined three industrial
scenarios for production of biologics:
1500, 500, and 150 kg/yr. The analysis
identified those steps with the greatest
impact on overall economics, allowing
outcome optimization.
Future of bioprocessing
Biopharma and, more specifically,
the field of bioprocessing are highly
conservative industries due to the stronglevel of regulation which can result inlong delays in introducing new technologies. Moreover, the inherent complexityof these activities increases the drag on
the process of innovation. Dan Kopec,
PAT technology expert at SSB, discussed
approaches to expediting technologicaladvances in bioprocessing.
The PAT (Process Analytical Technology) toolbox is, according to Kopec,“a system for designing, analyzing, andcontrolling bioprocess manufacturing.
This is accomplished through timely

measurements (i.e., during processing)of critical quality and performance attributes of raw and in-process materials
and processes. The overall goal is tominimize time to market and the costand risk of failure, while at the sametime, maximizing process productivity,efficiency, and flexibility.”
Kopec stressed the fact that today,
drug approvals move forward too
slowly, and the rate of approval is quite
low. According to a report by the Sloan
School of Business Management, only
14% of drugs in clinical trials eventually
win FDA approval. There is ample evidence that a more rapid and expeditious
system is badly needed in the bioprocessing industry, where a faster time and asurer path to market can be achieved
through better process understanding
and automation.
The application of the PAT strategy
can move bioprocessing from a rigid(fixed process) approach to one thatmaximizes cell specific productivity andproduct quality. An automated, flexible
feed strategy, which would be based on
a combination of real-time inputs fromPAT sensors. “This enables consistenthigh product quality,” Kopec stated.

Moreover, the integrated, dynamiccontrol provided by PAT allows a fast,predictive, and automated response tochanging conditions, also freeing up
operator time. These conditions can bemonitored by a combination of various forms of spectroscopy, including
NIR (Near-infrared spectroscopy), 2D
fluorescence, Raman. UV/Vis, scattering,and dedicated sensors like bio-capacitance, nutrient/metabolite and off-gas.
The benefits of this approach include
data and signal integration and real time
control.
“Using real-time sensors allows
a much clearer picture moving fromresearch to development to manufacturing,” said Kopec.
In looking to the future of nextgeneration manufacturing, Kopec statedthat some bioprocesses are challenging
to automate. These include existing
pipelines and related processes, and critical parameters/quality attributes thatlack available robust sensor technologies(e.g., differentiation between proteins).
“Ten years from now we anticipate
machine learning, fully automated processes and advanced simulation andprediction,” Kopec predicted.

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