Biochemical Technology (BIOT) Technical Sessions at the 253rd ACS Annual Meeting

1. Learn about
the latest innovations
Biochemical Technology (BIOT) Technical Sessions
at the 253rd ACS Annual Meeting
April 2-6, 2017 – San Francisco, CA
MilliporeSigma and our partners will be
presenting several technical presentations
and posters at the 253rd ACS National
Meeting & Exposition. If you are planning
to attend this event, please join our
experts who will be presenting the
latest developments in flocculation and
clarification, high viscosity TFF, vaccine
processing, performance optimization of
chromatography resins, high throughput
screening, Process Intensification and
continuous processing, and viral filtration.
The life science business of Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma in the U.S. and Canada.

2. Monday, April 3
82 - Linking single pass tangential
flow filtration with anion exchange
chromatography for intensified
mAb processing
10:30am - 10:50am
Downstream Processes - Disruptive
Bioprocessing & Process Integration
InterContinental B - InterContinental
San Francisco
Thomas Elich, Elizabeth Goodrich, Herb Lutz,
Ushma Mehta
MilliporeSigma, Billerica, Massachusetts,
United States
There is growing interest within the
biopharmaceutical industry to develop processes
which improve individual unit operation
performance and overall process train efficiency
through linked and continuous processing.
The use of single pass tangential flow filtration
(SPTFF) technology can help facilitate these
process intensification efforts by modifying
process intermediate volumes and concentrations
without the need for recirculation. In this work,
SPTFF is used prior to a flowthrough anion
exchange (AEX) mAb polishing step to boost
resin loading and productivity. Results are
presented which show that linking SPTFF with
AEX chromatography provides improvements
in both host cell protein impurity clearance and
process economics as compared to using AEX
alone. Excellent removal of MVM and XMuLV is
also maintained at the optimized conditions.
Considerations for developing and implementing
this approach as either a batch step or combined
with others as part of a continuous process are
also discussed.
Sunday, April 2
29 - Clarification by flocculation:
A promising solution for high cell density
7:00pm - 9:00pm
BIOL: Current Topics in Biochemistry
West Hall - Moscone Center
Clothilde Decarnin,
clothilde.decarnin@merckgroup.com
Merck, Schaffhausen, Switzerland
Advances in mammalian expression systems
have resulted in increased monoclonal antibody
titers link to high density cell cultures with
corresponding elevations in cellular debris
and cellular impurities. In such cases, the
performances of classical unit operations such
as centrifugation or depth filtration are
drastically reduced.
Although the effectiveness of flocculation has
been frequently demonstrated, adoption at large
scale is not yet common.
In this study from bench scale up to 2000 L
single-use bioreactor scale, we could implement
and witness the benefit of combining cell harvest
pretreatment with a polycationic flocculating
agent (polydiallyldimethylammonium chloride or
pDADMAC), followed by specific depth filters, able
to handle very large particle sizes (> 20 µm). This
robust process has not only allowed for reaching
higher filtration capacity and yield with enhanced
impurity removal, it has also exhibited its linear
scalability and great flexibility with limited foot
print and investment. We also demonstrate how
easy the implementation is as such scale, since
pre-treatment is done directly in the Bioreactor.
The area requirement of the subsequent depth
filtration is reduced compared to classical
depth filters.
Biological Chemistry (BIOL)
Technical Sessions at the
253rd ACS Annual Meeting

3. Tuesday, April 4
162 - High viscosity tangential flow
filtration applications: The impact of feed
channel screen on process performance,
high concentration capability, and
membrane re-use
10:30am - 10:50am
Downstream Processes
InterContinental B - InterContinental
San Francisco
Dana Kinzlmaier, Elizabeth Goodrich
MilliporeSigma, Billerica, Massachusetts,
United States
The current trends in biopharmaceutical industry
show increasing demand for high viscosity
tangential flow filtration (TFF) applications with
onset of novel drug delivery methods enabling
the administration of high concentration drug
products to patients. Traditional TFF devices
can be limited in reaching these concentrations
due to high pressures caused by highly viscous
feed streams. Filtration devices used during high
concentration processing have to be optimized
to handle both high viscosity and pressures
while maintaining high flux and excellent product
recovery. In this study, a family of filtration
devices was evaluated to characterize the impact
of membrane material and channel geometry on
process performance, viscosity capability, product
recovery, and cleanability when working with high
concentration protein streams. The results will
show the performance of each filtration device
over multiple re-uses, will present a solution
that can overcome process limitations due to
high viscosity formulations, and will demonstrate
the benefit of having a variety of feed channel
screen options to allow for optimization of process
performance across a range of applications.
182 - High throughput process development
application in the DoE study of insulin
analog capture step
8:50am - 9:10am
Downstream Processes
InterContinental B - InterContinental
San Francisco
Danny Wu (presenter), Siyi Liao, Bin Wang
MilliporeSigma, Billerica, Massachusetts,
United States
High Throughput Process Development (HTPD)
is central to the rapid optimization of biologics
purification processes. In this study, HTPD
strategies were applied in the DoE study of insulin
analog capture step. The study were divided into
three stages.
1) DBC evaluation. Loading pH and conductivity
were selected as input and evaluate the effect on
DBC with DoE approaches under HTPD platform.
2) Intermediate wash & elution optimization.
Intermediate wash pH, elution salt type and
elution salt concentration were selected as the
input factors in DoE study. Purity, Yield, Elution
peak residence time and Elution peak volume
were selected as responses and the optimization
zone was developed through HTPD platform;
3) Confirmation at larger scale. Scale-up
evaluation was performed under optimized
process conditions to compare the process
performance. The results were consistent with
the HTPD platform.
In conclusion, the application of HTPD in
chromatography optimization is a high efficiency
method which can achieve the similar result as
larger scale in a shorter time.

4. Tuesday, April 4
164 - Continuous in line flocculation, new
opportunities for primary separation and
economic impact
8:50am - 9:10am
Downstream Processes
InterContinental B - InterContinental
San Francisco
Peter Satzer2
, peter.satzer@boku.ac.at, Daniel
Burgstaller2
, Walpurga Krepper2
, Alessandro
Cataldo2
, Josselyn Haas3
, Marine Maszelin3
,
Jure Mohoric4
, Katja Pajnic4
, Alois Jungbauer1
1
Dept. Biotechnology, BOKU, Vienna, Austria
2
Biotechnology, University of Natural Resources
and Life Science, Vienna, Austria
3
Biomanufacturing Sciences Network, Merck,
Molsheim, France
4
LEK, a Sandoz company, Ljubljana, Slovenia
We present new approaches to flocculation
strategies for the removal of cells and impurities
during primary separation for mAb production.
The succeeded in reducing the necessary filtration
area during depth filtration of the harvest while
gaining additional benefits without adding
additional process steps. We tested three different
flocculation agents, a salt flocculation by Calcium
Chloride and polymer based flocculations using
Polydiallyldimethylammonium Chloride (pDADMAC)
or benzyl modified Polyallylamine (mPAA). All three
were combined with Clarisolve®
filters optimized
for flocculated harvest material. All three options
were successful in reducing the necessary filtration
area, the most beneficial effect showed pDADMAC.
On closer inspection each of the methods revealed
additional benefits besides the reduction of filtration
area. While pDADMAC and CaCl2 removed virtually
all DNA during flocculation given the right condition,
mPAA and CaCl2 were able to remove aggregates
with little yield losses. We followed the kinetics of
CaCl2 and pDADMAC flocculations by Focus Beam
Reflectance Measurements (FBRM) and video
microscopy. This data can be used if equilibrium
conditions are necessary for the further downstream,
or can be used to identify pre-steady-state conditions
beneficial to the filtration. We demonstrated the
suitability of in line continuous flocculation for batch
and for perfusion processes by parallelization of the
depth filtration. We continuously induced flocculation
in a feed stream, included a hold step of fixed length,
and the diverted the stream onto one of two filters.
Using in line flocculation opens up the opportunity of
using pre-steady-state flocculation conditions with
fixed time for floc evolution to optimize the filtration.
Using three scenarios, classical centrifugation,
depth filtration without flocculation and depth
filtration, sized and based on our lab scale
results, we evaluated the economic benefits of
in-line flocculation.
255 - mAb Aggregate removal using flow
through chromatography on fractogel
6:00pm - 8:00pm
BIOT Poster Session
West Hall - Moscone Center
Matthew Dillingham1
,
matthew.dillingham@merckgroup.com,
Ole Jensen2
, Nicolas Laroudie3
1
Technology Management, Millipore (UK) Ltd,
Edinburgh, United Kingdom
2
Novo Nordisk, Gentofte, Denmark
3
Manufacturing Sciences Technology, Merck,
Molsheim, France
Even though mAb platform processes are now
well developed and characterized, the propensity
for mAb aggregation and the need to remove
product related impurities can still be a bottleneck
and have economic repercussions both in process
cost and development time. This poster describes
the use of Fractogel®
COO- (weak cation
exchanger) to remove mAb aggregates in flow
through mode of operation. Previous work had
shown differences in selectivity for aggregates,
dimers and monomer in bind/elute mode, but
it was not possible to achieve the required
combination of purity and monomer recovery.
The flow through method presented here relies
upon adjustment of the feed conductivity to
maximize the selectivity between monomers
and aggregates/dimers. It was possible to
over–achieve customer requirements by
demonstrating 88% monomer recovery at a
loading of 200 g/L resin with removal of product
related impurities to the limit of the SEC detection
method. Scale up robustness was shown from
1 to 10 cm resin bed height. Flow through
purification methods are more easily adapted to
continuous processing and in this case achieved
a combination of purity, recovery and throughput
not possible in conventional bind/elute mode.
This technique makes highly efficient use of the
chromatography media (200 mg/ml gel) and the
absence of a high conductivity elution facilitates
subsequent polishing steps.

5. Tuesday, April 4
281 - Implementing single-use assemblies:
Design qualification testing implications
6:00pm - 8:00pm
BIOT Poster Session
West Hall - Moscone Center
Alexandra Steele,
alexandra.steele@emdmillipore.com
MilliporeSigma, Billerica, Massachusetts,
United States
The implementation of single use assemblies
in manufacturing spaces has raised concerns
around their proper functioning under process
conditions. This is highlighted in qualification
testing focusing on connections in final fill
assemblies where product leaks result in
high economic loss. The following case study
examines the implementation of bubble emission,
hydrostatic, and pressure decay tests to assess
for leaks and possible egress points on final fill
assemblies. These methods would be performed
as part of Design Qualification (DQ) testing.
DQ is performed once in the lifetime of an
assembly to provide confidence that the design,
included connections, tubing, fastenings, etc. will
withstand process conditions. Testing in the case
study was conducted on tubing engagements
using model assemblies containing 89 tubing,
hosebarb, and tri-clamp to tri-clamp (TC/TC)
connections and fastener permutations. Execution
of these tests during design qualification would
provide risk mitigation to help ensure the success
of single use assemblies in manufacturing.
339 - Long-term storage solution offering
membrane stability and bioburden
effectiveness for regenerated cellulose
ultrafiltration membrane cassettes
6:00pm - 8:00pm
BIOT Poster Session
West Hall - Moscone Center
William Cataldo, william.cataldo@emdmillipore.
com, Dennis Aquino, Mikhail Kozlov, Danielle
DeCesaro, Kathleen Souza, Andrew Bartlett
MilliporeSigma, Bedford, Massachusetts,
United States
Ultrafiltration (UF) membranes in cassettes
operating in Tangential Flow Filtration (TFF) mode
are the biopharmaceutical industry standard for
the concentration and diafiltration (UF/DF) of
mAbs, vaccines, peptides, and other therapeutic
proteins. Regenerated Cellulose (RC) composite
ultrafiltration membranes such as Ultracel®
(MilliporeSigma) are class leading UF membranes
that combine ultra-low protein binding, low
fouling, and organic solvent resistance with
superb mechanical strength. Ultracel®
is available
in Pellicon®
2 3 flat sheet TFF cassettes in
various molecular weight cut-offs (NMWCO)
for target molecule UF/DF processes. UF/DF
operations using cassettes in TFF mode offer high
retention, efficiency, flexibility, linear scalability,
and cost-effective reuse over several batch
operations. Typical end use of cassettes involves
a series of steps including flush-out, rinsing,
cleaning, sanitization, rinsing, and equilibration
before use. In order to be reused, cassettes must
be cleaned and filled with a storage solution
to ensure the membrane remains wet and to
prevent microbiological growth without damage
to the filter until next use. Devices can then be
flushed, cleaned, and sanitized prior to next use.
Some current recommended storage solutions
include 0.1 N NaOH, 0.1% Lysol, or 1600-2400
ppm Benzalkonium chloride (BAK), but only
guarantee 1 year stability and may present
flushing challenges. This work will present a new
long-term low pH buffer storage solution for
RC membrane cassettes. Data will show that it
provides 3-year membrane and cassette stability,
bioburden effectiveness, and low and benign
solute flush-out. Membrane and cassette stability
are demonstrated through no significant changes
in flux, dextran sieving, protein retention, and
device integrity. Bioburden effectiveness was
demonstrated through meeting USP 51
category 1 criteria for antimicrobial effectiveness.
Finally, flush-out data will show the low levels of
the common acid and base buffer components
used in this benign and effective long-term RC
cassette storage solution.

6. Tuesday, April 4
340 - Evaluating the correlation between
packing quality and process performance in
protein A chromatography
6:00pm - 8:00pm
BIOT Poster Session
West Hall - Moscone Center
Alejandro Becerra-Arteaga, Juan Castano,
juan.castano@emdmillipore.com
MilliporeSigma, Billerica, Massachusetts,
United States
The integrity of packed bed chromatography
columns is commonly evaluated to determine
if a column is acceptable to use for processing
a drug product intermediate. The integrity test
is performed under conditions in which the
packing factor in van Deemter’s equation is
maximized and the quality of the packed bed
is assessed by calculating the Asymmetry (As)
and Height Equivalent to a Theoretical Plate
(HETP). This same approach is used irrespective
of chromatography type, although the packing
factor is negligible in applications such as Protein
A affinity. During process development, lab scale
Protein A columns are rarely evaluated for HETP
or As, but a failure of either one of these values
during large scale column qualification leads to
resource intensive repacking operations. In this
work we evaluated the critical process parameters
(capacity, yield, purity, elution pool volume) for
Protein A columns having a broad range of As and
HETP including some outside the range of typical
acceptable values. The output of this work could
be leveraged to broaden the acceptance criteria
for Protein A column qualification and to reduce
the need to repack process-scale columns.
359 - Adapting single-use mixing solutions
for high titer drug product formulation:
A case study
6:00pm - 8:00pm
BIOT Poster Session
West Hall - Moscone Center
Adam Sokolnicki,
adam.sokolnicki@emdmillipore.com
Manufacturing Sciences Technology,
MilliporeSigma, Billerica, Massachusetts,
United States
Single-use mixing provides an agile adaptable
solution when bringing drug product formulation
steps in-house, as the trend towards higher titer,
smaller batches continues. Higher viscosities
associated with subcutaneous formulations
challenge the envisioned design space for
systems intended for gentle protein mixing. To
mitigate the risk to the process of suboptimal
mixing, this study evaluated the mixer design
space through blending studies with surrogate
solutions and with CFD modeling. Implementation
of these techniques helps to transform drug
product development and tech transfer strategies.

7. Tuesday, April 4
419 - Microcarrier removal using gradient
depth filters: Influenza vaccine case study
6:00pm - 8:00pm
BIOT Poster Session
West Hall - Moscone Center
Akshat Gupta1
, akshat.gupta.tech@gmail.com,
Sonal Patel2
, Lori Mullin2
, Meghan Higson2
,
Christopher Gillespie2
1
Applications Engineering, EMD Millipore
Corporation, Tewksbury, Massachusetts,
United States
2
EMD Millipore, Billerica, Massachusetts,
United States
Microcarrier based cell culture process is
becoming a preferred method of production for
a wide variety of biopharmaceutical modalities
such as recombinant proteins, vaccines and gene
therapy. The primary motivations for adopting
microcarrier based cell culture processes include
high productivity, scalability and utilization
of existing upstream infrastructure. Removal
of microcarriers during harvesting can be a
challenging task and current methods have
limited scalability. Traditional clarification
filters offer low microcarrier loading capacities
and tangential flow microfiltration processes
are capital equipment intensive and involve
tedious cleaning validation. In this work we
have demonstrated implementation of novel
gradient synthetic depth filters for microcarrier
removal. A case study for microcarrier removal
using gradient depth filters for cell based
Influenza was performed. The effects of various
process parameters including concentration of
microcarriers and loading flux on gradient depth
filter capacities were investigated. Additionally
secondary clarification optimization and sterile
filter sizing for influenza was carried out to
develop a complete clarification train. The new
approach offers greater than 40% increase in
loading capacity over traditional normal-flow
filtration. A successful 10 x scale up of the bench
scale process was carried out to demonstrate
linear scalability.
451 - Fundamental toolbox for protein A
resin characterization and link to application
efficiency
6:00pm - 8:00pm
BIOT Poster Session
West Hall - Moscone Center
Oliver Rammo, oliver.rammo@merckgroup.com,
Peter Menstell, Bianca Edelmann, Romas Skudas
Merck KGaA, Darmstadt, Germany
Protein-protein based interaction, regardless
its complexity, gained the biggest recognition
in biopharmaceutical industry. Especially the
discovery of Protein A and antibody interaction
enabled to reduce the complexity of this
pharmaceutical molecule group purification
resulting in 5 blockbuster drugs within the top 10.
And there are more to come taking into account
more than 2500 clinical studies ongoing.
Regardless the robustness of this method,
where remains an opened question how is this
protein-protein interaction taking place and what
governs the efficiency of the process if the Protein
A molecule is bound to the surface of the support
having limited accessibility.
We approached this challenge from user
perspective, using chromatographic performance
as a main criteria. Within our research we used
the combined power of mass spectroscopy,
quantitative amino acid analysis and
thermogravimetric and standard protein analytics
such as BCA/oPA and SDS-Page to distinguish
the immobilized Protein A, the number and
origin of domains and the linkers in between
them. This was followed by detecting the way of
immobilization (e.g. local of multiple attachment
zones) with the trace analysis of immobilized
amino acid or traces thereof. Finally we added
up some support physical characteristics with
confocal laser scanning microscopy (CLSM)
followed by the chromatographic performance for
different test molecules and operation modes.
In the presentation we will discuss the outcome
of our study, concentrating on the efficiency of
the chromatographic separation governed by
the enhanced diffusion (e.g. outcome of physical
properties from base support) and diversity of
possible protein-protein interaction, originating
from different Protein A domains. And finally we
will come back to our question: “The fundamental
toolbox for Protein A resin characterization and
link to application efficiency?”

8. Tuesday, April 4
453 - Case analysis of orthogonal, sterile
antibody purification platform
6:00pm - 8:00pm
BIOT Poster Session
West Hall - Moscone Center
Romas Skudas, romas.skudas@merckgroup.com,
Oliver Rammo, Peter Menstell
Merck KGaA, Darmstadt, Germany
There is a constant drive in biopharmaceutical
industry to increase the performance and economic
efficiency of the target molecule purification
maintaining the product quality attributes.
However, since the biopharmaceutical industry
is conservative, due to the intense regulatory
oversight, it is more attractive to seek for the
effective ways of using existing technologies rather
than risking early adoption of new technology with
little regulatory precedent.
One of those effective ways is the implementation
of no-bioburden continuous manufacturing
platform, based on the sterile Protein A capture
followed by orthogonal flow through polishing
principle. The orthogonal polishing principle is
a mixture of size exclusion, ion exchange and
hydrophobicity mechanisms.
Throughout several research years, we enabled
to establish a robust, reliable and low bioburden
antibody purification platform, assuring the
high purity standards of this technology in
biopharmaceutical industry.
To prove this, we applied gamma irradiation
at 25-40kGy or autoclaving at 121°C for 20
minutes for all used technologies in antibody
purification platform and performed case analysis,
enabling to reduce the HCP and DNA levels
to FDA requirements. Physical resin diffusion
characteristics were investigated with confocal
laser scanning microscopy (CLSM), showing no
radical change in adsorption performance.
5 case analysis were performed enabling the
low molecular weight reduction in 3 cases. HCP
10ppm was achieved in all 5 cases. Surprisingly,
in all cases the antibody recovery was 80%.
We believe that our finding will simplify and
increase the robustness of antibody purification,
enabling to maintain high product yields and
effective reduction of impurities in sterilized
platform without complex process development.
456 - Rapid and reliable analytical RP
HPLC for high-throughput insulin process
development
6:00pm - 8:00pm
BIOT Poster Session
West Hall - Moscone Center
Andreas Stein, andreas.stein@merckgroup.com,
Anja Heinen-Kreuzig, Andre Kiesewetter
Life Science, Merck KGaA, Darmstadt, Germany
The biopharmaceutical industry is strongly
moving to high-throughput formats for process
development. Powerful analytics are required as
an essential and integral part of this approach
and must deliver short analysis time, appropriate
resolution, efficient data handling, and flexible
output options.
Evolving from an 80 minutes routine protocol,
we have established a Reversed phase (RP) HPLC
method with a run time of only 8 minutes as a
rapid in-process analytical tool to support
high-throughput insulin purification development,
where 100 to 300 samples per day may be
generated from resin screening trials. The short
method, which uses a Chromolith®
HighResolution
RP-18 endcapped 100-4.6 column, maintains
adequate selectivity in the relevant region of
separation of the target insulin form and closely
related impurities, such as the des-amido insulin
molecule. We will demonstrate the comparability
of both methods with purity and titer data from a
range of process samples.
With a second focus on method reliability,
we have assessed and optimized the
evaluation of analytical chromatograms.
Blank subtraction, peak integration methods,
peak integration window, and linearity of peak
signals were all identified as crucial for accuracy
and reproducibility.

9. Tuesday, April 4
500 - TFF Optimization for small
molecule processing
6:00pm - 8:00pm
BIOT Poster Session
West Hall - Moscone Center
Emily Peterson, emily.peterson@live.com
MSAT, MilliporeSigma, Pepperell, Massachusetts,
United States
Due to their higher osmotic pressures and
mass transfer coefficients small molecules less
than 10 kDa, like insulin, often require unique
processing conditions as compared to those
of larger molecules. TFF processing strategies
developed for larger molecule applications may
not be appropriate and can lead to an increase in
process variability and sub-optimal performance.
This talk explores the key limitations and
challenges typically observed with small molecule
TFF processing and explains the strategies
required for optimal success with your TFF step.
578 - pDADMAC flocculation clarification
of CHO cell culture harvest
6:00pm - 8:00pm
BIOT Poster Session
West Hall - Moscone Center
Akshat Gupta1
, akshat.gupta.tech@gmail.com,
Juan Castano2
, Kara Pizzelli2
, Shannon Ryan2
1
Applications Engineering, EMD Millipore
Corporation, Tewksbury, Massachusetts,
United States
2
EMD Millipore, Billerica, Massachusetts,
United States
Over the last decade, contributions from both
academia and industry have significantly
enhanced the knowledge base on poly (di allyl
di methyl ammonium chloride) (pDADMAC)
flocculation for CHO cell culture harvesting. In the
current work we continue to fill existing gaps in
process development best practices and provide
insight on scale up and GMP implementation
aspects of this technology. In this study we
investigated the impact of different methods of
mixing, mixing speeds and method of flocculant
addition on particle size distribution and
clarification capacity. Scalability of the clarification
process using gradient depth media from bench
to process scale was demonstrated for pDADMAC
flocculated CHO cell culture fluid. Another key
aspect for GMP implementation is successful
cleaning validation. In this presentation data for
cleanability of stainless steel surfaces soiled
with pDADMAC flocculated cell culture will also
be presented.

10. Thursday, April 6
680 - Effect of product concentration on
continuous parvovirus-retentive filtration
10:30am - 10:50am
Downstream Processes - Advances in
Non-Chromatographic Separations
InterContinental B - InterContinental
San Francisco
David Bohonak and Herb Lutz
MilliporeSigma, Billerica, Massachusetts,
United States
Use of higher product concentrations in process
intermediate streams offers opportunities to
intensify downstream processes and facilitate
adoption of more continuous approaches to
biomanufacturing. However, there is limited
fundamental understanding of how increased
concentrations may affect parvovirus-retentive
filtration, even though this step has a significant
effect on manufacturing costs. In this work,
experimental studies were conducted to
characterize the performance of adsorptive
membrane prefilters commonly used to enhance
virus filter capacity for a range of concentrations.
In addition to varying the total protein
concentration, the concentration of large product
aggregates was varied independently. These large
aggregates are typically responsible for fouling
of virus filters and limit their capacity, and their
adsorption was compared to common adsorption
isotherms. The effects of protein and aggregate
concentrations on the virus filter permeability
and capacity were studied separately to quantify
the contributions of solution viscosity, particulate
fouling, and other phenomena. These results
were coupled with filter sizing studies under
various flux conditions specific to continuous
processing requirements and with optimization of
the feed pH and conductivity to demonstrate a set
of strategies to minimize filter area requirements
and replacement frequency.
681 - Investigation of flushing strategies for
reducing Beta Glucan Leachables originating
from cellulose based depth filtration media
10:50am - 11:10am
Downstream Processes
InterContinental C - InterContinental
San Francisco
Akshat Gupta, Dana Kinzlmaier, Kara Pizzelli and
Elizabeth Goodrich
MilliporeSigma, Billerica, Massachusetts,
United States
Depth filters composed of cellulose and
diatomaceous earth can be used at multiple
stages in a monoclonal antibody purification
process for removal of various cellular impurities.
The cellulosic component of these filters is
known to contain trace levels of β-(1-3) Glucan
impurities which can potentially leach into the
product stream during processing. As leached
β-(1-3) Glucan are considered a process impurity,
levels need to be maintained below a defined
threshold as per ICH Q6B guidance. In this work
different flushing strategies were investigated to
reduce the β-(1-3) Glucan leachables originating
from cellulosic depth media. Effect of various
process parameters including buffer formulation,
method of contacting, and flush volume to area
ratios were investigated. Effect of these flushes
on depth filter performance was also investigated.
An optimized specialized flushing strategy offers
up to 80 % reduction in β-(1-3) Glucan leachable
levels with no change in filter performance.

11. Thursday, April 6
710 - Evaluation of a new all-synthetic
depth filter media demonstrates improved
clarification and soluble impurity clearance
post-protein A and low pH viral inactivation
2:40pm - 11:00pm
Downstream Processes
InterContinental C - InterContinental
San Francisco
Hoang Nguyen1
, hank.nguyen1@gmail.com,
Amie Langland1
, Brooke Meyer1
, David Kahn1
,
Joseph Costanzo1
, John Amara2
1
Virology and Purification Development,
Eli Lilly and Company, Indianapolis, Indiana,
United States
2
Clarification, MilliporeSigma, Bedford,
Massachusetts, United States
Studies were conducted to evaluate a new,
all-synthetic depth filtration media comprised of
silica and a charged polymeric component. The
evaluation sought to demonstrate the enhanced
ability of the new media to clarify post-Protein
A, post low pH viral inactivation precipitate and
soluble impurities (e.g. aggregate, host cell
proteins (HCPs), residual Protein A) compared
to benchmark cellulosic media. Representative
proteins derived from mammalian cell culture
were tested at two pH conditions. The studies
additionally investigated the effects of clarification
and soluble impurity clearance when specific
depth filter characteristics were varied across
their manufacturing specification ranges (e.g.
%Silica, Layer Thickness, Water Permeability).
In general, the new media was superior in
performance to the existing benchmarks,
specifically in reducing soluble impurities such
as HCPs and aggregate, however in some cases
reduced filter loads (g/m2
) were observed. No
discernable differences in impurity clearance
or filter loads were observed when filter
specifications were varied. However, increased
silica leaching in certain filtrate samples was
observed for the new filters, but forward
processing that material through downstream
polishing chromatography columns demonstrated
adequate silica clearance.

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