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14 Δεκ 2013 (πριν από 3 χρόνια και 6 μήνες)

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Prep 2010 Abstract/Ahmed (11
-
18
-
09)


ABSTRACT # 001


Preference:

Oral


Presenter:


Hasnat Ahmed

Ph.D student

Institute of Chemistry

Karl Franzens
University
Graz

Heinrichstrasse 28,

A
-
8010 Graz,
AUSTRIA

hasnatmalik47@yahoo.com



Preparative Scale
Fractionation of Poly(
ε
-
caprolactone
)

Block Copolymers and Their Identification by
Liquid Chromatography and MALDI
-
TOF
-
MS.
Ha
s
nat

A
hm
e
d
,
B
e
r
nd

T
r
athnigg,
C
.

Oli
ve
r
K
ap
p
e
,

R
ob
e
r
t

S
a
f,
Institute of Chemistry, Karl Franzens
University
Graz, Heinrichstrasse
28, A
-
8010 Graz, AUSTRIA


Aliphatic polyesters belong to a class of biodegradable polymers which have applications in
pharmaceutical and biomedical engineering. Among the polyesters poly (ε
-
caprolactone) (PCL) is one of
the most promising materials
.
Its
applicatios are however limited because of its hydrophobicity and high
crystallinity
. However on introduction of polyethylene

glycol (PEG) chains can transform them to
amphiphilic polymers whose properties can be manipulated by adjusting the ratio of hydro
philic and
hydrophobic components
[1,2]
.

We have synthesize their diblock and triblock copolymers with
monofunctional and difunctional PEG initiators respectively
[3,4]
. These polymers were fractionated using
semi preparative Spherisorb ODS2 (25 x 300 mm, 5


m, 80 Å) column with 2 ml/min flow rate at 25
o
C.
The system was equiped with 500 ul loop and density detector was used for all measurements. Sample
concentrations were 3.0 to 20.0 g/l. Fractionations were carried out under critical conditions for ethyle
ne
oxide repeat unit using acetonitril:water as the mobile phase. Under such conditions separation was
made on the basis of adsorption of CL units. These fractions were then subjected to different analytical
columns and MALDI
-
TOF
-
MS for their identificatio
n and qunatification. By using this analytical approach
we were able to develope the methods for separation of homopolymers, undesired cycles, block
copolymers and even their discrimnation to di and triblock copolymers. [1] Kumar N, Ravikumar MNV,
Domb AJ,

Advanced Drug Delivery Reviews 2001;53:23
-
44. [2] ZhangC, Wen X, Vyavahare NR, Boland
T, Biomaterials 2008; 29:3781
-
3791. [3] Ciardelli G, Rechichi A, Cerrai P, Tricoli M, Giusti P,
Macromolecular Symposia 2004; 218:261
-
272. [4] Ahmed H, Trathnigg B, Oliv
er Kappe C, Saf R, Eur
Polym J 2009;45:2338
-
2347.





Prep 2010 Abstract/Carta (11
-
19
-
09)








ABSTRACT # 002



Preference:

Oral

Please note that I will likely have to depart by Tuesday noon


Presenter:


Giorgio Carta

Department of Chemical Engineering

University of Virginia

102 Engineers' Way

Charlottesville, VA 22904
-
4741

Phone:

434
-
924
-
6281

FAX: 434
-
982
-
2658


gc@cms.mail.virginia.edu




pH Transients in Hydroxyapatite Columns
-

Chemistry and Dynamic Modeling.
Theresa Bankston, Laura
Dattolo,
Giorgio Carta
, Department of Chemical Engineering, University of Virginia, Charlottesville,
VA
22904
-
4742, USA


Hydroxyapatite columns are widely used for chromatographic separation of proteins and other
biomolecules becaus
e of their unique selectivity and ability to resolve complex mixtures. Separation
occurs because of selective binding to negatively charged phosphate surface groups (refereed to as P
-
sites), selective binding to positively charged surface calcium sites (re
ferred to as C
-
sites), or through a
combination of both. Design and operation of these columns require special care since hydroxyapatite
exhibits a finite aqueous solubility al low pH. Thus, operation above pH 6 is generally required. A
significant complic
ation, however, is that unintended pH transients can occur in hydroxyapatite columns
when the salt concentration varies. For example, the pH temporarily decreases below the feed value
when the salt concentration increases and increases above the feed value

when the salt concentration is
increased, and this occurs even when using suitably buffered solutions. The intensity and duration of
these transients depends on the particular buffer used and the magnitude of the salt concentration step,
but in extreme ca
ses the pH can drop by as much as 2 pH units creating conditions where stability of the
hydroxyapatite is compromised. Thus, it is critical to be able to predict under what conditions this can
occur. In this work we have examined in detail the mechanism le
ading to pH transients in CHT Type I
columns generated by salt steps with phosphate buffers at pH 6.5. The pH excursions are similar to those
observed for weak cation exchange columns, but are accompanied by a transient evolution of phosphate
which decreas
es below the feed value when the salt concentration is increased and increases sharply
when the salt concentration is reduced. The interplay of salt and phosphate waves results in complex pH
patterns with a sudden pH drop followed by an intermediate state
before returning to the feed pH values
when the column is subject to a positive salt concentration step. A model is developed to describe these
waves by considering the reversible uptake of sodium ions by the P
-
sites and the competitive binding of
monovale
nt and divalent phosphate ions on the C
-
sites. The latter is only weakly dependent on the salt
concentration but is strongly affected by pH, which, in turn, varies when sodium ions are adsorbed or
released from the hydroxyapatite surface. The interplay of
these two interrelated adsorption mechanisms
results in complex waves that are consistent with those observed experimentally. In addition to helping
understand the underlying mechanisms, the model also provides a useful tool to predict the effects of
diffe
rent buffers and salt concentration and develop corrective measures that can reduce the intensity and
duration of the pH transients.





Prep 2010 Abstract/Mao (11
-
16
-
09)








ABSTRACT # 003


Preference:


Oral


Presenter:


Nathan Mao, Ph.D.

Senior Engine
er, BioPharm Develop

Biogen Idec

5200 Research Pl.

San Diego, CA 92122

Tel: 858
-
401
-
5146

Fax: 858
-
795
-
9289

nathan.mao@biogenidec.com



Leveraging Downstream Depth Filtration for HCP Removal in a Two
-
column Purification Process of MAb.
Nathan Mao
, Yik Lam,
Matthew Westoby, John Pieracci, Process Biochemistry, Biogen IDEC, San Diego,
CA 92122, USA


In this case study, post protein
-
A depth filtration was used to enhance the clearance removal of a
standard two
-
column purification process. The depth filters were

determined to be capable of significant
HCP clearance (up to 2.1 logs) without which implementation of a two column process might not be
feasible. Since the mechanism and robustness of HCP clearance by depth filtration was not well
understood, factorial d
esign studies were performed to identify the significant parameters affecting HCP
clearance. The optimal operating conditions were located and the robustness of the process was
established within the experimental space. By combining harvest step conducted
at low pH and post
protein
-
A column depth filtration, the desired HCP clearance was achieved with a single chromatography
column process. The final recommended process included a membrane adsorbent to insure robust viral
clearance claims.




Prep 2010 Abs
tract/Black


ABSTRACT#

004


Preference:
Oral


Presenter:

Regina Black

Merck and Co.



Strategic Use of Semipreparative Supercritical Fluid Chromatography (SFC) to Enable Pharmaceutical
Lead Optimization and Early Development. Regina Black, Preparative Ch
emistry and Separat
ions, Merck
and Co., Rahway, NJ, USA


Supercritical fluid chromatography (SFC) has become an established tool in the pharmaceutical industry
for fast, labor
-
efficient and green purifications in support of discovery and development resear
ch. As the
technique of preparative SFC continues to evolve, and as synthetic chemists become more comfortable
with the technique, new opportunities for incorporating preparative SFC into synthetic strategy continue to
be identified and exploited. In thi
s presentation, several recent examples from our laboratories will be
presented that illustrate the increasingly important role that semi
-
preparative SFC plays in the initial
synthesis and early development of pharmaceutical development candidates, with pa
rticular emphasis on
examples where separation scientists collaborate with synthetic chemists to develop new strategies to
address increasingly complex synthetic challenges.




PREP 2010 Abstract/Silva (12
-
1
-
09)


ABSTRACT#

005


Preference:
Oral


Presenter:

Ricardo JS Silva


Submitted by:

José Paulo Mota

Associate Professor

Requimte/CQFB

Chemistry Department

FCT/UNL

2829
-
516 Caparica
, PORTUGAL

Tel.:

+351212948385

pmota@dq.fct.unl.pt



Measurement and Analysis of Cooperative Adsorption Isotherms of Binaphtho
l Enantiomers on
Chiralpack AD.
Ricardo J. S. Silva
,

Rui C. R. Rodrigues, José P. B. Mota, Requimte/CQFB,
D
epartamento de Química, Faculdade de Cências e Tecnologia, Universidade Nova de Lisboa, 2829
-
516
Caparica,
PORTUGAL


The cooperative adsorption of
solutes is an unusual phenomenon and has been hardly reported. Such
interaction between solutes is suggested by the results of the present study, where the adsorption
isotherms of the isolated enantiomers of binaphtol, and those of the racemic mixture, wer
e measured by
Frontal Analysis by Characteristic Point, using a mixture of 2
-
propanol and n
-
hexane as mobile phase
and Chiralpack AD as stationary phase. The experimental data obtained for the individual solutes show
that the most
-
retained solute exhibits
an inflexion point in its adsorption isotherm, whereas the less
-
retained solute exhibits a nearly linear adsorption isotherm. However, when both solutes are present in
solution, a cooperative interaction between them is clearly observed in the individual a
dsorption
isotherms.

To account for the physical observations in the adsorption behaviour of the most retained
solute, two adsorption isotherm models are proposed and compared: an adsorption isotherm model
based on statistical thermodynamics and an adsorpt
ion isotherm model derived from the Virial equation.
The cooperative phenomena of the adsorption of both solutes on the chiral stationary phase can be
explained by the adsorption of a complex formed by both solutes present in solution, where the total
adso
rption isotherm for each solute is a sum of two effects: the individual solute adsorption and the
adsorption of the complex formed.

Keywords:

adsorption isotherms, chiral stationary phase, frontal
analysis, cooperative adsorption






Prep 2010 Abstract/Ka
spereit


ABSTRACT#

006


Preference:
Oral


Presenter:

Malte Kaspereit

Max Planck Institute for Dynamics of Complex Technical Systems


Sandtorstr. 1, D
-
39106 Magdeburg /
GERMANY


phone: +49 391 6110
-

282, fax:
-

551


kaspereit@mpi
-
magdeburg.mpg.de



Steady
State Recycling Chromatography


Simplified Optimal Design and Options for Process
Improvement.
Malte Kaspereit
1
,

Tuomo

Sainio

2
, Achim Kienle
1,3
,
1
Max Planck Institute for Dynamics of
Complex Technical Systems, Magdeburg,
GERMANY;
2
Laboratory of Industria
l Chemistry, Lappeenranta
University of Technology, Lappeenranta,
FINLAND;
3
Otto
-
von
-
Guericke University Magdeburg, Chair for
Automation/Modeling, Magdeburg,
GERMANY


Several single
-
column techniques exist that aim at improving elution chromatography by re
cycling the
chromatogram partially or as a whole. Among them is the pseudo
-
continuous steady state recycling
(SSR) chromatography. This concept gains increasing interest for binary separations, because its lower
investment costs can make it an interesting
alternative to simulated moving bed chromatography.

The
SSR concept is based on collecting the purified leading and trailing parts of the elution profile, and the
addition of a constant amount of fresh feed to the unresolved recycle fractions. After a suff
icient number
of injections, the process attains a so
-
called periodic steady state. Optimal design and analysis of SSR
separations are difficult. Recently we developed a design method that

greatly simplifies these tasks
[1]
.
The approach works for
user
-
defined purity or yield requirements and holds for negligible mass transfer
limitations. For such systems it was proved that the SSR concept always achieves better eluent
consumption and product concentrations, but a lower productivity than an optimiz
ed batch process. The
method also predicts the steady state. This allows eliminating completely the startup period of the
process.

In the present work, we extend the approach to cases where mass transfer resistances cannot
be neglected. We develop a simple

shortcut design method that requires as input only a single
overloaded injection. The approach is validated theoretically and experimentally for the separation of two
cycleketones on a hydrophobic polystyrene resin. We show that, for systems with limited
column
efficiency, the SSR concept achieves not only higher product concentrations and lower eluent
consumption, but also higher productivity than batch chromatography.

Finally, different options are
discussed for further improvement of the SSR concept. Th
is includes the optimized combination of SSR
systems with isomerization reactions and integration of membrane
-
based solvent removal. Corresponding
experiments are currently performed for an industrially relevant chiral separation.

Acknowledgement
:
This
wor
k is supported by the European Commission under the grant FP7
-
NMP2
-
SL2008
-
214129 (project
INTENANT


“Integrated synthesis and purification of single enantiomers”)
.

[1]

Sainio, T., Kaspereit, M.,
Analysis of steady state recycling chromat
ography using equilibrium theory, Sep. Purif. Technol. 66
(2009) 9

18



Feed

(E1, E2)

PREP 2010 Abstract/Rodrigues (12
-
1
-
09)


ABSTRACT#

007


Preference:
Oral


Presenter:

Rui CR Rodrigues


Submitted by:

José Paulo Mota

Associate Professor

Requimte/CQFB

Chemistry
Department

FCT/UNL

2829
-
516 Caparica
, PORTUGAL

Tel.:

+351212948385

pmota@dq.fct.unl.pt


Comparison of Streamlined, Two
-
Column, Simulated Moving
-
Bed Processes for Chiral Separation.
Rui C.
R. Rodrigues
, Ricardo J. S. Silva,

José P. B. Mota, Requimte/CQFB, Departamento de Química,
Faculdade de Cências e Tecnologia, Universidade Nova de Lisboa, 2829
-
516 Caparica,
PORTUGAL


Preparative and process
-
scale chromatography have been used for decades in the pharmaceutical and
fine che
mistry industries, for the separation and purification of a wide range of substances. These
industries use mostly single
-
column batch chromatography, except for some more recent multi
-
column
applications


notably simulated moving
-
bed (SMB) processes

, an
d are somewhat skeptical about
changing to multi
-
column, continuous chromatography. This is, in part, because such a change may
represent not only a big step from economical and operational viewpoints, but also increased complexity
in terms of process vali
dation. However, since in pharmaceutics and fine chemistry the annual volume of
processed products is low when compared to petrochemical or sugar industries, there is room for simple,
compact, and easy to operate and validate, yet efficient, small
-
scale ch
romatographic processes.

In the present work we compare the performances of two streamlined two
-
column chromatographic
processes for binary separation and batch chromatography with ideal recycle


an idealized version of the
steady
-
state recycling (SSR) te
chnique where the time delay and extra
-
column dispersion originated by
the injection loop are absent). The streamlined processes combine a flexible node design


to exploit the
benefits of simulated counter
-
current adsorption


and simplicity of physical r
ealization: regardless of the
number of columns, only two pumps are needed to supply feed and desorbent into the system, while the
flow rates of liquid withdrawn from the system are controlled by material balance using simple two
-
way
valves. The two stream
lined processes supply fresh feed into the system where the composition of the
circulating fluid is closest to that of the feedstock fluid, and recover the product


either extract or raffinate


in purified form at the downstream end of the unit while des
orbent is supplied into the upstream end of
the system. One of the designs employs an internal recirculation pump to implement a recirculation step
where the inlet and outlet ports are closed and the internal flow through the system moves the
concentration

profiles along the two columns to adjust their relative position with respect to the outlet
ports. The feasibility and effectiveness of the two
-
column process are verified and compared
experimentally on the separation of reboxetine racemate


a norepineph
rine re
-
uptake inhibitor


under
overloaded conditions, and on the linear separation of nucleosides by reversed phase.

Keywords:

two
-
column processes, simulated moving bed, chiral separation



PREP 2010 Abstract/Kostareva


ABSTRACT# 008


Preference:
Oral


Presenter:

Irina Kostareva, PhD

Business Development Manager,

Technical Support Specialist

ATOLL
-
Bio USA Inc.

P.O.Box 630171, Riverdale, NY 10463

Phone: 718 884
-
2080

Cell: 917 566
-
0783

t.schroeder@atoll
-
bio.
com



Parallel Chromatography in 96
-
Well Formatted MiniColumns in Multi
-
Step Protein Purification Process
Development.
Tim Schroeder
, Jürgen Friedle, Atoll GmbH, Ettishofer Straße 10, D
-
88250 Weingarten,
Germany, Irina Kostareva, Atoll
-
Bio USA, P.O.Box
630171, Riverdale, NY 10463
, USA



The evaluation and optimization of chromatographic conditions is a time consuming task in the
development of processes for the purification of therapeutic proteins. MiniColumns in a 96
-
well format are
used to speed up the

selection of resin and purification parameters by means of either automated or
manual HTP screening technology. The MAb polishing anion exchange step was developed by loading
Protein A eluate onto 8 PipetColumns packed with different AIEX media (positive
liquid displacement
pipette flow). Using the flow
-
through method, the best AEIX resin was selected. Purification conditions
were optimized by running eight RoboColumns packed with the chosen AEIX resin in parallel using an
automated liquid handler. Proce
ss scale
-
up to 10 ml confirmed the results obtained using 50 µl
MiniColumns. Atoll MediaScout HTP screening technology combined with the automated Freedom EVO
Tecan Liquid handling workstation accelerated purification process development by nearly one orde
r of
magnitude.



Can we do both, oral and poster presentation.

Atoll has developed a new technology platform to obtain results in much faster time frames, almost 10
times, due to the use of robotics for parallel chromatography.

This new technique is of
very high interest to the biopharm industry for screening, process control and
drug discovery tasks. Many of these companies are now looking into an investment for robotic stations.



An oral presentation decribes the technique as such and

various applicat
ion areas, whereas the poster
will describe in more detail a certain application e.g. in process development.




Please, Janet, advice us what needs to be done to have both, oral and poster presentation.


12
-
1
-
09


advised her to submit a poster abstract,
titles must differ.

And again 2
-
18
-
10



PREP 2010 Abstract/Mota (12
-
1
-
09)


ABSTRACT#

009


Preference:
Oral


Presenter:

José Paulo Mota

Associate Professor

Requimte/CQFB

Chemistry Department

FCT/UNL

2829
-
516 Caparica
, PORTUGAL

Tel.:

+351212948385

pmota@dq.fct.unl.pt



A New Multicolumn, Open
-
Loop Process For Ternary Separation By Solvent
-
Gradient Chromatography.
José P. B. Mota
,
1*

Rui C. R. Rodrigues,
1

Ricardo J. S. Silva
1
,
H. Osuna
-
Sanchez,
2

E. Valéry
2
,
1
Requimte/CQFB, Departamento de Química,
Faculda
de de Cências e Tecnologia, Uni
versidade Nova
de Lisboa, 2829
-
516 Caparica,
PORTUGAL;
2
N
ova
S
ep
Process, Boulevard de la Moselle, BP 50
-

54340
Pompey,
FRANCE


For many biopurification problems, the desired product is intermediate between weakly and
strongly
adsorbing impurities, and a central cut is thus required to get the desired product.

We report results on
the proof of concept of a new process (Gradient Steady State Recycling
-

GSSR)
recently developed. The
GSSR process comprises a multi
-
column,

open
-
loop system with a solvent gradient and cyclic steady
-
state operation. It is particularly suited for ternary separations: it provides three main fractions or products,
with a target product contained in the intermediate fraction.

A comprehensive
description of the GSSR
process is given, highlighting the versatility, flexibility, and ease of operation of the process. We provide
experimental validation in a pilot unit, using the purification of a crude peptide mixture by reversed phase
as a benchmar
k and case study. This crude mixture has ca. 50% purity and contains impurities with
retention times very close to the target component, which makes it a challenging separation by
conventional batch column chromatography even when incorporating solvent gra
dients. The entire unit is
fully automated and controlled using in
-
house developed software that manages the automatic updating
of the port switching, valve positions, pump flow rates, and automatic sampling. Experimental results are
reported in terms of c
yclic steady
-
state profiles and process performance indicators: product purity, yield,
productivity and solvent consumption. A model
-
based approach is employed to explain the behavior of
the process. We have been able to get pure product that meets our tar
get specifications


98% purity
and 95% recovery


under stable operation.

Keywords:

biochromatography, center
-
cut separation,
solvent gradient, multi
-
column process, open
-
loop configuration



PREP2010 ABSTRACT/Hessler (11
-
30
-
09) approved 2
-
4
-
10


ABSTRACT
#

010


Preference:

Oral


Presenter:


Andrew Hessler

GlaxoSmithKline

Analytical Sciences, Chemical Development

709 Swedeland Road

King of Prussia, PA 19406

610
-
270
-
5566

andrew.j.hessler@gsk.com

CC:
xiqin.2.yang@gsk.com
,
Leo.Hsu@gsk.com
,
David.W.Thornton@gsk
.com



Rescue Chromatography: Development of a Purification Process to Recover an Intermediate from Mother
Liquor.

Andrew Hessler
, Xiqin Yang, David Thornton, Leo Hsu, Chemical Development, GlaxoSmithKline,
709 Swedeland Road, King of Prussia, PA 19406, US
A


There are several approaches to produce chemically pure drug substances including recrystallization,
distillation, and direct chromatographic resolution. In most production scale processes, recrystallization is
the first choice of technology to remove
impurities. However, when the yield from the recrystallization
process is low, preparative chromatography can be used to recover material from mother liquor. We
developed a preparative chromatography method to recover an intermediate from mother liquor
co
ntaining N
-
methyl
-
2
-
pyrrolidone (NMP) and benzyl alcohol. Detailed method development using various
chromatographic techniques will also be presented.










PREP2010 ABSTRACT/Knight (12
-
1
-
09)


ABSTRACT #

011


Preference:

Oral


Presenter:

Martha Knight

CC Biotech LLC

9700 Great Seneca Highway

Rockville, MD 20850
-
3307

marthaknight@ccbiotech.us



Application of Spiral Countercurrent Chromatography to
Peptides and
Proteins.
Martha Knight
1
, Thomas
M. Finn
1
, Adam Clayton
2
, John Zehmer
2
,
1
CC Biotech LLC and
2
APC Biotechnology Services, Inc., 9700
Great Seneca Highway, Rockville, MD 20850
-
3307, USA


Spiral countercurrent chromatography (CCC) is a liquid
-
liquid partitioning method using flowthrough
spiral
-
nested tubing separation rotors operated in planetary cen
trifuges. The previously reported spiral
tubing support (STS) rotor has been applied to protein separations using the aqueous two phase solvent
systems (ATPS). We will show results of using polyethylene glycol (PEG)
-
phosphate salts
-
solvent
systems to separ
ate mixtures of small proteins and mixtures of larger proteins. The strategy of solvent
system selection from the growing field of protein extraction will be described. One
-
step isolation of
proteins out of expression system lysates is a goal and experimen
ts to achieve this will be presented. The
spiral CCC method has good loading capacity which makes it able to provide useful preparative
separations in the research lab.





PREP2010 ABSTRACT/Kraettli (11
-
26
-
09)


ABSTRACT #

012


Preference:

Oral


Presenter:


Martin Krättli

ETH Zurich

Institute for Chemical
-

and Bioengineering (ICB)

Morbidelli Group, HCI F 122

Wolfgang Pauli Strasse 10

8093 Zurich, SWITZERLAND

Phone: +41 (0)44 632 56 88

martin.kraettli@chem.ethz.ch

www.morbidelli
-
group.ethz.ch



Cycle to Cycle Open Loop Control in Protein Purification Applying Continuous Chromatography
(MCSGP).
Martin

Krättli
1
, Guido Ströhlein
1,2
, Lars Aumann
1,2
, Thomas Müller
-
Späth
1,2
, Massimo
Morbidelli
1
,
1
Institute of Chemical and Bioengineering, Department of Chemistry and Applied Bioscience,
ETH Zurich, CH
-
8093 Zürich, SWITZERLAND;
2
ChromaCon AG, Technoparkstr. 1,

CH
-
8005 Zürich,
SWITZERLAND


Continuous chromatography based on the multicolumn countercurrent solvent gradient purification
(MCSGP) technology has the potential to purify proteins with high yield and purity, e. g. in the field of
monoclonal antibody (mAb
) purification
1

and mAb variant separation
2

using preparative ion exchange
chromatography. In order to reduce the number of prestudies for process design and to increase the
robustness of the MCSGP process against disturbances during operation, a control c
oncept was
developed. Recently, a cycle to cycle control concept for simulated moving bed chromatography (SMB)
based on analytical HPLC measurements of the product streams was elaborated
3
. In this work a control
concept for MCSGP was developed and verified

experimentally using only online UV measurements
obtained throughout the cycle at each column outlet. The UV signal of all columns is averaged and used
as feedback for the controller. According to this, an optimized cycle time is determined. Advantages an
d
disadvantages evolving from this control concept will be discussed and compared to other approaches.
For a model separation, a procedure will be shown how to design and control the MCSGP process from
one overloaded batch chromatography experiment. Refere
nces: (1) Aumann, Morbidelli, ”A

semicontinuous 3
-
column countercurrent solvent gradient purification (MCSGP) process”, Biotechnology
and Bioengineering 2008; 99(3): 728


732. (2) Müller
-
Späth, et al., “Chromatographic separation of three
monoclonal antib
ody variants using multicolumn countercurrent solvent gradient purification (MCSGP)“,
Biotechnology and Bioengineering 2008; 100(6): 1166


1177. (3) Langel, et al., “
Implementation of an
automated on
-
line high
-
performance liquid chromatography monitoring
system for ‘cycle to cycle’ control
of simulated moving beds
”, Journal of Chromatography A 2009; 1216(50): 8806
-

8815




PREP2010 ABSTRACT/Jermann (12
-
2
-
09)


ABSTRACT #

013


Preference:

Oral


Presenter:

Simon Jermann

sjermann@student.ethz.ch

CC:
marco.mazzotti@ipe.mavt.ethz.ch



Fast Development of a Chiral SMB Separation Under Nonlinear Chromatographic Conditions by On
-
line
Optimizing Control.
Simon Jermann
, Christian Langel, Cri
stian Grossmann, Marco Mazzotti
, Manfred
Morari, Massimo Morbidelli,

ETH Zurich, Zürich, SWITZERLAND


Simulated Moving Bed (SMB) chromatography has become one of the key techniques in pharmaceutical
industry to separate enantiomers and the fast adaptation of an existing SMB unit to a new separation task
has thus become mor
e important. Our group has reported on the experimental implementation of an
automated on
-
line HPLC monitoring system for ‘cycle to cycle’ control of simulated moving beds (SMB)
1
.
Since this control scheme requires only little information about the system,

which is easy to determine, a
given SMB unit can be rapidly set up for a new separation. This work presents the necessary steps
required to set up a new chiral SMB separation making use of our on
-
line optimizing control scheme and
shows that the developme
nt time is of only three to four days. This was shown on the separation of the
enantiomers Troeger’s base in ethanol using ChiralpakTM AD as stationary phase. After designing the
SMB separation, experiments with specified product purities of 98.5% and feed

concentrations of 0.75 g/l,
4.0 g/l and 8.0 g/l were run, i.e. the chromatographic conditions ranged from linear to considerably
nonlinear. However, when choosing the same initial operating point the dynamic behaviour of the
controller was identical for t
he different runs though the final operating points differed due to the
increasing nonlinearity. In addition to the experiments, rigorous simulations using the bi
-
Langmuir
isotherms determined in a previous work
2

were performed. This allowed for the first
time the direct
comparison of experimental and theoretical results. As the bi
-
Langmuir parameters used in the
simulations were determined for another batch of chromatographic columns the final operating points
differed slightly between experiments and simu
lations. However, the dynamic behaviour observed in the
experiments was exactly reflected in the simulations. The simulations further showed that the control
scheme is able to cope with feed concentrations close to the solubility limit and hence allows for

operation
with higher productivity. As far as changes during operation are concerned it was observed that the
controller rejected both unintentional and designed disturbances reliably, and it guaranteed the fulfilment
of the purity specifications under an
y circumstances. References: (1) C. Langel, C. Grossmann, M.
Morbidelli, M. Morari, M. Mazzotti,
J. Chrom. A
1216
(2009) 8806
-
8815. (2) S. Katsuo, M. Mazzotti,
J.
Chrom. A
(2009) submitted.





PREP2010 ABSTRACT/Kofoed
-
Hansen (12
-
2
-
09)


ABSTRACT #

014


Pre
ference:

Oral


Presenter:

Britt Kofoed
-
Hansen

Senior Application Chemist

Separation Products

Eka Chemicals AB

SE
-
445 80 Bohus, Sweden

T +46 31 58 7315

M +46 31 709 57 7315

F +46 31 58 7727

britt.kofoed
-
hansen@akzonobel.com



Case Study: Separation and Purification of Two Chiral Discovery Compounds
using Kromasil CSPs, The
Influence of Particle Size on Process Productivity in Chiral Batch Chromatography.
Joakim Högblom,
Sylvia Winkel Pettersson
,
Britt Kofoed
-
Hansen
,
Akzo Nobel,

Separation Products, SE
-
445 80 Bohus,
SWEDEN


Chiral chromatography is a well established and growing separation technique. The need to separate and
purify chiral compounds increases continuously within the pharmaceutical industry. HPLC is known to be
a
fast and efficient technique for chiral separations in every scale, but SFC is approaching from behind
promising faster, cheaper and more efficient purifications. This presentation outlines a case study of the
successful separation and purification of two
chiral leads. Different separation alternatives with respect to
stationary phase, mobile phase and chromatographic mode are identified. A number of the separation
alternatives are scaled up to preparative scale. The case study exemplifies how the efficienc
y given by
the particle size of the stationary phase is fully utilized in both HPLC and SFC mode. Additionally the
issue of sample solubility and mobile phase strength in relation to total process output is covered.
Separation characteristics suitable for
continuous chromatography were encountered and are discussed
in the presentation as well. The quick and feasible scale up of two chiral separations in drug discovery is
demonstrated and the economy of gram scale purification is calculated.





PREP2010 ABS
TRACT/Kreuss (11
-
23
-
09)


ABSTRACT #

015


Preference:

Oral


Presenter:

OLD

Dipl.
-
Ing. M.Sc Markus Kreuss

Lehrstuhl für Lebensmittelverfahrenstechnik

Weihenstephaner Berg 1

85354 Freising, GERMANY

Tel.: 0049
-
8161
-
715032

Fax: 0049
-
8161
-
714384

markus.kreuss@wzw.tum.de


new:

Markus Kreuss

Proteinchemie
-

Wirkstoffproduktion


Rentschler Biotechnologie GmbH

Erwin Rentschler Strasse 21

88471 Laupheim

T +49 (0) 7392 701 738

F +49 (0) 7392 701
-
825

mailto:Markus.Kreuss@rentschler.de

http://www.rentschler.de




Development of a Preparative Ion Exchange Process for the Separation of Glycosylated Whey Pepti
des
using Membrane Adsorption Chromatography.
Markus
Kreuss
a
,

Elena

Leeb
b
, Ulrich Kulozik
b
,
a
Rentschler
Biotechnologie, Erwin
-
Rentschler
-
Str. 21, D
-
88471 Laupheim, GERMANY;
b
Technische

Universität
München, Chair for Food Process Engineering and Dairy Technology, Weihenstephaner Berg 1, D
-
85354
Freising, GERMANY


The aim of this study was to develop and optimize an anionic membrane adsorption chromatography
(MAC) process for the pilot p
lant scale fractionation of the biological and technological active whey
peptide caseinomacropeptide (CMP) into its glycosylated (gCMP) and non
-
glycosylated (aCMP) fractions,
which are supposed to differ in biofunctionality. CMP has a rather heterogeneous
composition with
respect to different genetic variants and posttranslational modifications such as phosphorylation and
glycosylation. A sufficient knowledge regarding the separation and ongoing research on the technological
and bioactive potential of the g
lyco
-
/aglyco
-
fractions of CMP is still lacking. Further on, the fractionation
method was adopted for direct capture (without pre
-
filtration) of CMP or gCMP directly out of sweet whey
and the overall process was optimized and coupled to an UF
-
diafiltration
process for desalting of the
eluate. Processing conditions can be varied using different buffers, pH, ionic strengths, flow rates and
elution profiles. The main target was the optimization of binding of the gCMP on the adsorber as well as
the minimization
of the overall process time and consumption of chemicals. It was shown that CMP could
be separated in the two major fractions gCMP and aCMP using an anionic MAC device. Best process
parameters where achieved using a food compatible sodium acetate buffer (0
.02 M) at a pH of 4.2. The
loading of the sample on the direct
-
capture module is best done in a fast flow recirculation mode. After a
washing step in order to clear out unbound molecules from the module, the elution of the bound glyco
-
CMP was done by incre
asing the sodium chloride level up to 0.7 M. It was further shown that both the
loading and the elution of the sample could be done at highest flow rates (10

L/min, which is 10 times the
MAC module volume) without any loss of purity and recovery. The metho
d was evolved using a
commercial CMP isolate as feedstock as well as fresh sweet whey from skim milk. The former resulted in
a binding capacity (BC) of 0.28

mg gCMP

/

cm² membrane surface with a purity of 97% while the latter
afforded a gCMP fraction with
a purity of 91% and a BC of 0.21

mg gCMP

/

cm² membrane surface. The
main difference was a significant fouling of the membrane adsorber module when the whey was applied,
which resulted in a loss of 46% BC after at least five loading/elution cycles. This ef
fect was not observed
using the pure CMP isolates and indicates a blocking of the ion
-
exchange ligands. This fouling can largely
be related to blocking of the ligands within the membrane pores according to the internal fouling model as
well as triglyceride
s of the whey. The fouling was decreased using microfiltered whey or by increasing the
temperature of the adsorption process. Additionally, a method of repeated elution was shown to decrease
the volume of the eluate as well as the NaCl consumption of the e
lution buffer. Keywords: membrane
adsorption chromatography, ion exchange chromatography, caseinomacropeptide, whey proteins.




PREP2010 ABSTRACT/Ferreira (12
-
2
-
09)


ABSTRACT #

016


Preference:

Oral (?)


Presenter:


Guilherme Ferreira


IBB
-
Institute for
Biotechnology and Bioengineering

Centre for Molecular and Structural Biomedicine

Universidade do Algarve

Faro, PORTUGAL

gferrei@ualg.pt



Thick Shear Mode Acoustic Devices to Study Biomolec
ular Recognition and Protein Binding.
Luis F. M.
Rosa, Jorge Carvalho,
Rogério Rodrigues
,
Guilherme N. M. Ferreira
,
IBB
-
Institute for Biotechnology and
Bioengineering, Centre for Molecular and Structural Biomedicine, Universidade do Algarve, Faro,
PORTUGAL


Acoustic wave sensors have been shown to be highly effective functional devices to monitor biomolecular
binding in real time. The thickness shear mode (TSM) acoustic wave sensor is the most popular acoustic
wave device, generally known as Quartz Crystal
Microbalance (QCM). Upon real time monitoring the
variation of the sensor resonance frequency and motional resistance, or dissipation factor, and using
acoustic impedance spectroscopy to model equivalent electric circuits the acoustic wave signal can be
cl
eared up by distinguishing mass load from acoustic energy losses and charge induced parasite
capacitive interferences due to the liquid viscosity and the presence of electrolytes, respectively. We have
previously shown that this strategy was highly effecti
ve to assess both equilibrium and kinetic constants
while giving some incites regarding conformational alterations and hydrophobicity of adsorbed proteins at
the sensor surface. In this work we extent the methodology and use acoustic impedance to screen
bi
nding of proteins to modified sensor surfaces. The goal is to establish specific correlations between
acoustic energy losses and protein hydrophobicity as well as to directly monitor eventual conformational
alterations upon protein binding.
Funding is ackn
owledged from IBB/CBME, LA, FEDER/POCI 2010 and
project PTDC/BIO/72307/2006.





PREP2010 ABSTRACT/Grabski (12
-
1
-
09)


ABSTRACT #

017


Preference:

Oral


Presenter:

Anthony C. Grabski, Ph.D.

Director of R&D

Semba Biosciences, Inc.

505 S. Rosa Road

Madison,
WI 53719

T 608.441.8061

Fax 608.441.8329

tgrabski@sembabio.com

www.sembabio.com


Continuous Protein Purification by Simulated Moving Bed: An Open Platform for Increased Productivity
and Purity.
Anthony G
rabski
, Robert Mierendorf, Semba Biosciences, Inc., 505 South Rosa Road,
Madison, WI 53719, USA


Simulated moving bed chromatography (SMBC), has been successfully applied to small and large
-
scale
binary separations of hydrocarbons, sugars, and enantiomers
but has rarely been used for protein
purifications. SMBC emulates counter current separation where the buffers and feed stream flow in the
opposite direction of the chromatography columns. Valves between the columns successively switched
open or closed at
timed intervals introduce the inlet streams and withdraw the outlet streams. Stepwise
advance of these streams in the direction of fluid flow simulates counter current movement of the
columns. The highly efficient zones of separation created by SMBC enable

continuous bind
-
wash
-
elute
chromatography and peak shaving of high purity target molecule. A bench
-
scale SMBC instrument was
employed to investigate several purification methods including Protein A, Protein G and immobilized
metal affinity purifications.
Advantages of SMBC over standard linear or batch processes include
dramatically increased productivity, purity, and efficiencies. These advantages can help alleviate the
bioprocessing bottleneck created by increasingly higher titers of protein production u
pstream making
SMBC an improved process alternative to single column methods.





PREP2010 ABSTRACT/Guelat (12
-
1
-
09)


ABSTRACT #

018



Preference:

Oral


Presenter:

Tarafder presenting

Bertrand Guélat, M.Sc.

ETH Zurich

Institute for Chemical and Bioengine
ering

Morbidelli Group

HCI F 136

Wolfgang
-
Pauli
-
Strasse 10

8093 Zurich, SWITZERLAND

bertrand.guelat@chem.ethz.ch

www.morbidelli.ethz.ch

+41 44 63

37

696 phone

+41 44 63 21 082 fax



Non
-
linear
Isotherm with pH and Salt Dependence for Protein Adsorption in Ion
-
exchange
Chromatography.
Bertrand Guélat
1
, Guido Ströhlein
1,2
, Thomas Müller
-
Späth
1,2
, Marco Lattuada
1
,
Abhijit
Tarafder
1
, Massimo Morbidelli
1
,
1
Institute for Chemical and Bioengineering, ETH Zurich, 8093 Zürich,
SWITZERLAND;
2
ChromaCon AG, Technoparkstr. 1, 8005 Zürich, SWITZERLAND


A fundamental understanding and modeling of protein adsorption in ion
-
exchange (IEX) chromatography
is desirable. Su
ch a model can serve as the basis for rational process design and characterization. In
process design, significant efforts are put in the substitution of the classical 3
-
steps purification process
(including expensive Protein
-
A capture) with a 2
-
steps proc
ess based on non
-
affinity chromatography, in
particular using ion
-
exchange chromatography. In this case, the challenging separations require detailed
optimizations which can be efficiently performed
in silico
, provided that reliable models are available. T
his
modeling approach would be also useful in the characterization and validation of the purification process.
A better process understanding is the basis to characterize the effect of the process variability (crude
concentration, aggregates, operating con
ditions, etc.) on the final product. Furthermore, such a model
complies with the concept of “Quality by Design” and could be used in the approval procedure following
the new regulation guidelines (ICH Q8). In this work, a model focused on the effect of the

pH and of the
salt concentration on the adsorption behavior of proteins is developed. Those two parameters are the
main operating conditions used to control the adsorption (i.e. retention time, selectivity) in ion
-
exchange
chromatography. First, the adsor
ption equilibrium has been modeled by computing the protein
-
surface
interactions from the electrostatic double
-
layer theory
1
. In a second step, the mechanism of resin
saturation by adsorbed proteins will be studied. In particular, the effect of protein
-
pro
tein interactions with
the increasing surface concentration will be modeled. The effect of the pH and of the salt concentration
on both the adsorption equilibrium and the saturation capacity will be explicitly accounted for. The
isotherm model will be expe
rimentally verified through the effect of the adsorption equilibrium and of the
saturation capacity on the retention time and peak shape of overloaded elutions. Model proteins and
industrial monoclonal antibodies will be used in this verification. Referenc
e: (1) B. Guélat, G. Ströhlein, T.
Müller
-
Späth, M. Lattuada, A. Tarafder and M. Morbidelli; “pH and salt dependence of protein adsorption
in ion
-
exchange chromatography”, PREP 2009
-

22
nd

International Symposium, Philadelphia, PA, USA.
Keywords: Adsorptio
n isotherm, monoclonal antibodies, process optimization, Quality by Design.




PREP2010 ABSTRACT/Fraud (11
-
11
-
09)


ABSTRACT #

019


Preference:

Oral


Presenter:

Nathalie Fraud, Ph.D.

Senior Scientist

Sartorius Stedim Biotech

Purification Process Developm
ent

1922 Santa Rosa Ave

Pasadena CA 91104

faPhone: +1.626.241.2171

Fax +1.626.389.0575

nathalie.fraud@sartorius
-
stedim.com



Salt Tolerant Membrane Adsorber for Robust Contaminant Removal.
Nathalie Fraud
1
, Rene Faber
2
,
Yujing Yang
1
, Uwe Gottschalk
2
,
1
Sa
rtorius Stedim North America, Bohemia, NY 11716, USA;
2
Sartorius
Stedim Biotech GmbH, D
-
37079 Goettingen, GERMANY



Anion Exchange Chromatography (AEX) is a commonly used process in the purification of monoclonal
antibodies. Because the isoelectric points
(p
I
’s) of many MAb’s occur at relatively high pH values
compared to the contaminants, AIEX are typically used as a polishing step in product flow
-
through mode.
Due to the convective mode of mass transport coupled with the need for only trace levels of impu
rity
clearance, Q
-
membrane chromatography involves much smaller devices than traditional Q
-
resin with
similar output. The separation is conducted at near neutral pH and low conductivity because high salt
levels disrupt electrostatic interactions, reducing
or eliminating the binding of the impurities onto the Q
ligand. For robust contaminant removal even

at high salt concentration, a new membrane adsorber
(Sartobind STIC) was developed. The Sartobind STIC allows polishing to be carried out without an
interst
itial dilution step, which reduces process time and avoids additional buffer preparation and hold
steps. Properties and performance of the new Sartobind STIC is presented.







PREP2010 ABSTRACT/Gronberg (12
-
1
-
09)


ABSTRACT #

020


Preference:

Oral


Presenter:


Anna Grönberg

Sr. Research Engineer, R&D

GE Healthcare Bio
-
Sciences AB

Björkgatan 30

SE
-
751 84 Uppsala,
SWEDEN

Anna.Gronberg@ge.com

CC: Hans.J.Johansson@ge.com



High
-
throu
ghput Screening of Cleaning
-
in
-
place Protocols for Antibody and Antibody Fragment
Purification on Affinity Chromatography Resins.
Anna Grönberg
, Enrique Carredano, Kjell Eriksson, Anna
Mjärdestam, Susanne Nyholm Westin, Hans J. Johansson,
GE Healthcare Bio
-
Sciences AB, Björkgatan
30, SE
-
751 84 Uppsala, SWEDEN


Design of efficient and resin compatible cleaning
-
in
-
place (CIP) protocols are essential for long term use
of chromatography resins in biopharmaceutical production. Traditional column life
-
time studie
s are time
and material consuming, and restricted to evaluation of one cleaning protocol at a time. Therefore, a high
-
throughput method was developed where 96
-
well filter plates filled with chromatography resin were used
for parallel evaluation of CIP proc
edures. The resins in the wells were fouled with challenging mammalian
and bacterial feed stocks, as well as human plasma. The cleaning efficiency of a large number of different
chemicals and sequences of cleaning steps were evaluated by analyzing the resi
dual amount of protein
on the beads after cleaning. Efficient CIP protocols were identified based on the assumption that an
efficient CIP protocol will result in a clean bead. The throughput of the screening method was maximized
by using fully automated ro
botic systems for plate handling and high
-
throughput analysis for quantification
of residual impurities on the resin. Finally, identified CIP protocols were evaluated in column format
through normal cycling, where target protein and impurity carry
-
over wer
e measured in periodic mock
eluates from blank cycles. Here, cleaning procedures were designed for affinity resins using antibody
containing feeds, but the screening method is applicable for design of CIP protocols for any combination
of feed and chromatog
raphy resin. The correlation between the scale
-
down screening format and
traditional life time studies in columns will be discussed. Process economy calculations comparing
different resins and cleaning regimes will also be presented.








PREP2010 ABSTRA
CT/Collier (11
-
24
-
09)


ABSTRACT #

021


Preference:

Poster


Submitter:


Jo
-
Ann_Jablonski@waters.com


Presenter:

Steven M. Collier

Waters Corporation

34 Maple Street

Milford, MA 01757

Phone: 800
-
252
-
4752, Extension 2158

steven_m_collier@waters.com



Methods
for Improving Synthetic Peptide Isolation. Jo
-
Ann M. Jablonski, Thomas E. Wheat, Kenneth J.
Fountain,
Steven M. Collier
, Waters Corporation, 34 Maple Street, Milford, MA 01757, USA


Peptides have many biological functions and are essential for the research

and development of
biopharmaceuticals. Even though the synthesis and cleavage of the peptide is carefully controlled,
numerous impurities are generated. Impurities include deleted and truncated sequences, cleavage
adducts, incomplete deprotections, and mo
dified amino acids. All of these contaminants must be
removed from the target peptide for unambiguous results in future experiments. Several approaches are
available to adjust the purification process for improved yield of pure material. Some peptide mixtu
res are
difficult to dissolve and keep in solution throughout the isolation process. Solvents like dimethylformamide
or dimethylsulfoxide are good for dissolving samples but their use can jeopardize the chromatographic
purification. The patented at
-
column
dilution technique, used in conjunction with temperature control and
focused gradients, improves chromatographic resolution, column mass capacity, and purification system
ruggedness by preventing sample precipitation. Controlling the temperature of the iso
lation at the large
scale vastly improves the purification of the product by improving the peptide’s solubility in the mobile
phase and increasing efficiency. Focused gradients give better resolution of the peptide product from its
closely
-
eluting contamin
ants without increasing run time. In this study, we illustrate the use of at
-
column
dilution, temperature control and focused gradients for improving synthetic peptide isolation. Employing
these techniques ultimately lead to improved process efficiency and

peptide products with higher purity
and increased yield.




PREP2010 ABSTRACT/deNeuville (11
-
26
-
09)


ABSTRACT #

022


Preference:

Oral


Presenter:


Bertrand de Neuville

ETH Zürich

Institute for Chemical and Bioengineering

Morbidelli group

HCI F 122

Wolfgang
-
Pauli Strasse 10

8093 Zürich, Switzerland


+41 44 633 68 60

bertrand.deneuville@chem.ethz.ch

www.morbidelli
-
group.ethz.ch





Effect of Pore Shrinkage on Macromolecular Diffusion in Porous Beads.
Bertrand de Neuville
, A.
Tarafder, H. Thomas, M. Morbidelli, Institute for Chemical
-

and Bioengineering,ETH Zürich,
Wolfgang
-
Pauli
-
Str. 10, CH
-
8093 Zürich, SWITZERLAND


Modeling of chromatographic separation of proteins, especially antibodies, needs special consideration
because their radius is close to the average size of the particle pores of the chromatographic media
.
Consequently, they cannot have access to all the pores and their adsorption on the pore walls can
critically modify the general pore accessibility.
This pore shrinkage also influences strongly the intra
-
particle diffusion that is usually the rate
-
limitin
g step and which ultimately controls the performance of
such processes.

So, a better understanding of the mass transport and adsorption mechanism inside the
pores is vital for designing efficient separation processes for antibodies.
In this direction, the
diffusion and
adsorption phenomena were studied at the pore scale using a model representing the pore size
distribution of the particles by a set of seven different pore diameters. In the model, the intra
-
particle
diffusion is considered as dependent on th
e pore diameter and also on the local amount of adsorbed
proteins. To investigate the transport behaviour of macromolecules, like Dextrans of different sizes, the
model was first characterised with results obtained from ISEC experiments. The variation in p
ore
accessibility and transport behaviour was then studied for various columns, e.g. ion
-
exchange columns
with tentacles; and with various process conditions, e.g. columns saturated with IgG molecules at
different salt concentrations. The outcomes of these

studies were then verified with corresponding
experimental results, carried out in the said conditions. The studies generated completely new insight
about the individual roles of the pores of different sizes, influence of molecular size on the transport
m
echanisms, pore blockage by adsorbed molecules and of the adsorption process at the pore scale.





PREP2010 ABSTRACT/Cox (12
-
1
-
09)


ABSTRACT #

023


Preference:

Oral


Presenter:


Geoffrey B Cox, Ph.D.

VP Technology

Chiral Technologies Inc.

800 North Five
Points Rd.

West Chester, PA 19380

(610) 594 2100 ext 234


gcox@chiraltech.com




Practical Aspects of Preparative Enantioselective Supercritical Fluid Chromatography. Screening and
Scale
-
up using Immobilized Polysaccharide Chiral Stationary Phases and Non
-
alcohol
-
based Modifiers.
Geoffrey B. Cox
, Bruce Coryell, James Lee, William Watts, Chiral Technologies, Inc., 800 N. Five Points
Road, West Chester, PA 19380, USA


Polysaccharide chiral stationary phases continue to predominate as the preferred media for
enantioselective preparative SFC. Use of coated
-
type stationary phases places limits on the mobile
phase modifiers that can be safely used without incurring some degree of damage to the stationary phase


either by slow degradation or catastrophic failure,

depending on the nature and concentration of the
modifier concerned. While SFC is in some ways more forgiving of excursions away from the traditional
alcohol modifiers than is HPLC, it does not guarantee safe operation of such columns under all
conditions
. The difficulty is that often the samples are not especially soluble in the alcohol mobile phase
modifiers and chromatographers are continually searching for solvents which can be used to enhance
solubility and production rate in SFC.

We have previously p
resented the effects of the mobile phase
modifier on selectivity
1

and loading
2

in preparative chiral SFC using several immobilized polysaccharide
CSPs and many “non
-
traditional” modifiers. The present work builds upon this base in developing a
screening an
d loading strategy for the development of suitable preparative SFC methods using a small
set of model compounds. The predicted loadings for each stationary phase


mobile phase combination
are confirmed using semi
-
preparative scale separations with 21 and
30 mm id columns and the
separations are optimized using a variety of column lengths to maximize the production rate. The
influence of the nature and composition of the mobile phase modifier on the product recovery is another
practical question in preparat
ive SFC, as condensation and precipitation of the more volatile modifiers
can potentially be problematic. The study reports on the recovery of modifier with and without the
presence of additional modifier components such as alcohols (often used to adjust t
he mobile phase
polarity) as well as upon the recovery of products from the separations investigated. References: (1)
Solvent and stationary phase selectivity in enantioselective SFC, G. B. Cox, SFC 2007, Pittsburgh, PA.
(2) Optimization strategies for fas
t preparative enantioselective SFC separations, Geoffrey B. Cox, Brian
Krueger, James K. Lee and Norbert Maier, PREP 2008, San Jose, CA.




PREP2010 ABSTRACT/Eriksson (12
-
1
-
09)


ABSTRACT #

024


Preference:

Oral


Presenter:

Kjell Eriksson

Ph D, Senior
Scientist

GE Healthcare

Life Science R&D

Bjorkgatan 30

751 84 Uppsala,
SWEDEN

T +46 (0)18 6120539

M +46 (0)70 6121027

kjell.eriksson@ge.com

www.gehealthcare.com



Rapid Method Development for MAb Purification from Challenging Feed.
Kjell Eriksson
, Anders Ljunglöf
and Tuomo Frigård, GE Healthcare Bio
-
Sciences, Björkgatan 30, SE
-
751 84 Uppsala, SWEDEN



A typical MAb purification processes norma
lly includes 3 chromatographic purification steps. The high
purity obtained after capture on protein A medium, i.e. MabSelect SuRe (typically > 99%), in combination
with the multimodal functionality of the ion exchange medium Capto adhere, allows reduction

of the
number of steps normally performed to a highly productive two
-
step process. The polishing step using
Capto adhere is preferably done in flow through mode under conditions that allow antibodies to pass
directly through a column while contaminants ar
e adsorbed. As always loading conditions will be a
compromise between those favoring yield and those favoring contaminant clearance, much so in this
case where several contaminant species needs to be removed. Loading conditions therefore need to be
optimiz
ed, and there will be a trade
-
off between yield and purity. Using a newly developed
chromatography instrument designed for process development we demonstrated a rapid method
development of a two step process for MAb purification from a challenging feed bas
ed on a clarified CHO
cell supernatant. The method development included determination of elution
-
pH and dynamic binding
capacity on MabSelect SuRe, and screening of loading conditions on Capto adhere using pH
-
elution
experiment followed by a robustness
-

an
d verification study. The screening and robustness study was
done using design of experiments (DoE).






PREP2010 ABSTRACT/Emde (
12
-
1
-
09
)


ABSTRACT #

025


Preference:

Oral


Presenter:


Dr. Ulrich Emde

Merck KGaA, Darmstadt

Merck Serono

MS
-
RTC
-
MLF2

Frankfurter Str. 250

D
-
64293 Darmstadt

Germany

ulrich.emde@merck.de

phone ++ 49 (0)6151 72 4018

fax ++ 49 (0)6151 72 3129



Preparative Liquid Chromatography for Compound Purification and Separation in Medicinal Chemistry.
Ulrich Emde
, Melanie Dietz, Dieter Spuck, Merck KGaA, Darmstadt
; Merck Serono, MS
-
RTC
-
MLF2,
Frankfurter Str. 250, D
-
64293 Darmstadt, GERMANY


Today in medicinal chemistry laboratories all over the world preparative liquid chromatography in various
forms (e.g. normal phase or revered phase, CLC or HPLC/SFC) is the most

used tool for compound
purification or separation. This talk shares some insights in this field seen from the viewpoint of a
preparative chromatographic unit inside of a medicinal chemistry department. The use of preparative LC
-
MS for rapid compound purif
ication is described. Our basic purification philosophy, column selection and
some interesting purification examples will be highlighted. Separation of all kind of stereoisomers is
another major business. Here we will lay out our general chiral column scre
ening strategy and show
some interesting case studies and observations (e.g. on atropisomerism) we made during the last seven
years. In the field of stereoisomer separations supercritical fluid chromatography (SFC) has become a
first class choice. Our five

years of experience will be summarized. Last but not least the recent set up of
a preparative SFC unit is described including comments on carbon dioxide supply, safety issues and how
to avoid potential pit falls during the installation.







PREP2010
ABSTRACT/Abrar (11
-
30
-
09)


ABSTRACT #

026


Preference:

POSTER


Presenter:

Shazia Abrar

PhD Student

Institute Of Chemistry

Karl
-
Franzens University of Graz

A
-
8010 Graz, Austria

43
-
69910744317

shazia.abrar@edu.uni
-
graz.at

bernd.trathnigg@uni
-
graz.at


Characterization of Commercial Polymeric Surfactants by Offline Two Dimensional Liquid
Chromatography.
Shazia Abrar (Mag)
, Bernd Trathnigg,
Central Polymer Lab,
Institute of Chemistry, Karl
-
Franzens University of

Graz,

Heinrichstraße 28, A
-
8010 Graz, AUSTRIA


Sugar based surfactants are important class of amphiphilic materials. Commercial polymeric surfactants
have a very complex composition due to presence of difference functionalities in the mixture of surfacta
nt
materials. High performance liquid chromatography is an efficient tool for the separation and
characterization of complex polymeric materials
1
. Different modes of liquid chromatography have been
used for the characterization of complex structured materi
al. For the complete characterization of
hetergenous samples one
-
dimensional separation does not reveal the structural complexity. Two
-
dimensional liquid chromatography has been used efficiently for the structure determination of the
complex synthetic poly
mers. Liquid adsorption chromatography under critical conditions of the repeating
units gives separation of different ester functionalities
1,2
. Using preparative conditions of the separations,
different functionalities have been separated from each other.
The collected fractions are further
subjected to other modes of liquid chromatography and spectroscopic techniques to get the complete
information about the architecture of the separated materials
3
. Preparative Spherclone ODS and
Spherisorb ODS columns hav
e been used for these separations. Different ester functionalities that were
separated are then further studies for their structure determinations. Comprehensive Two
-
dimensional
approach for the samples under study does not reveal a very good information f
or the complete structural
analysis due to incompatibility of different separation conditions i.e. mobile phase compositions and
separation. Mono, di and higher esters are separated under the reverse phase separation conditions.
However all the peaks could

not be assigned for their functionality due to presence of different side
products in the sample composition. Matrix Assisted Lasar Desorption Time of Flight Mass Spectrometry
confirmed the separated functionalities for their structure. The separated frac
tions were also studies
under normal phase condition using Silica column for the separation according the length of their
polyethylene block. A complete picture about the structure of the complex surfactant samples was
obtained. Keywords: Polysorbate surfa
ctants, Preparative separations, liquid chromatography.
References:
[1] Matyjaszewski, K., Miller, P. J., Pyun, J., Kickelbick, G., Diamanti, S., Macromolecules,
1999, 32, 6526
-
6535.
[2] Im, K., Park, H.
-
w., Lee, S., Chang, T., Journal of Chro
matography A, 2009,
1216, 4606
-
4610. [3] Vu Dang, H., Gray, A. I., Watson, D., Bates, C. D., Scholes, P., Eccleston, G. M.,
Journal of Pharmaceutical and Biomedical Analysis, 2006, 40, 1155
-
1165.




PREP2010 ABSTRACT/Ahmad (12
-
1
-
09)


ABSTRACT #

027


Preference:

Oral


Presenter:

Tarab Ahmad

Western Illinois University

Department of Chemistry

324 Currens Hall

Macomb, IL 61455

309
-
298
-
1656

tj
-
ahmad@wiu.edu



Investigation of the Use of Room Temperature Ionic Liquids as Organic Mobile Phase Replacements for
Reversed
-
phase Liquid Chromatography.
Tarab Ahmad
a
, Divya Shekar
a
, Vijaya sree Vegesna
a
,
Prashanthi

Kolanupaka
a
, and Tariq Z. Ahmad
b
,
a
Department of Chemistry, Western Illinois University,
Macomb, IL, USA;
b
Macomb Senior High School, Macomb, IL, USA


Ionic liquids or room temperature ionic liquids RTILs are salts with melting points at or close to room

temperature. They are good solvents, highly polar, environmentally benign, nonvolatile, nonflammable,
and stable in air or water. Because of these particular properties, ionic liquids (named as ‘‘green
chemistry’’ solvents) are currently, of considerable

interest in the separation and analysis. RTILs are used
in analytical liquid chromatography either as mobile phase additives or as a replacement for organic
modifiers. Whether they are used as mobile phase additives or as a replacement for organic modifi
er,
RTILs can be used to adjust the selectivity as well as to enhance the chromatographic resolution by
improving the peak shapes. While further progress is required before ionic liquids become mainstream
analytical solvents, results to date commend their
use in various modes of chemical analysis. There has
been little research on ILs used in RP
-
HPLC, and the good properties of ILs have not been exploited
completely. In order to better understand the retention behavior of amino acids on RP
-
HPLC in the
prese
nce of RTIL we are carrying two types of investigations. First the adsorption behavior of some
amino acids on RP stationary phase is investigated using different 1,3 dialkyl
-
substituted imidazolium
salts (R: ethyl, butyl, and octyl) as mobile phase additi
ves. The effects of the chain length of the alkyl
substituent, the concentration of the ionic liquids and the type of counter ions on the adsorption behavior
of some amino acids on RP are studied. Second we are investigating adsorption behavior of the same

analytes on RP
-
HPLC using alkylammonium Formate (AAF) ionic liquids (R:

ethyl, propyl and butyl) as
organic mobile phase replacements. The Results of both investigations will be presented.





PREP2010 ABSTRACT/Borg (12
-
1
-
09)


ABSTRACT #

028


Preference
:

Poster


Presenter:


Niklas Borg

Department of Chemical Engineering

Lund University

P.O. Box 124

SE
-
221 00 Lund,
SWEDEN

NiklasBo@chemeng.lth.se



Model for Distributed Pore

Volumes.
Niklas Borg
, Karin Westerberg, Niklas Andersson, Bernt Nilsson,
Department of Chemical Engineering, Lund University, P.O. Box 124, SE
-
221 00 Lund, SWEDEN



As the concentrations of proteins increases in fermentation, the requirements of the downs
tream
processing are also increased. To meet these new requirements, a better understanding of the process is
needed. To easier be able to design a process based on a mathematical model, it is useful to have
different models to choose from. Derivatives can

be hard to remove in a capture column since their
properties are similar to that of the native target protein. [Kelley el al Biotech and Bioeng 101:3 (2008)
553] Size exclusion chromatography has been known to be used for this separation[Cutler], but the
development has been toward other modes of chromatography. The general rate model has been
successfully used to describe what happens inside the particles of those gels, but sometimes this is not
enough. To describe the consequence of size exclusion effect
s on a preparative adsorption column a
model has been implemented to separate volumes of the gel that are competitive for all components to
volumes of the gel that are only accessible for one component. The model has been designed in a way
that does not in
troduce more parameters than a normal kinetic
-
dispersive or general rate model and that
does not interfere with the model for the isotherm. The determination of the voids in the particles will get
an increased influence. The model is based on the assumptio
n that the ligand is homogeneously
distributed in the gel and that the different components can only access the ligand in parts of the gel
where the component is small enough to fit. The gel is then divided into parts where all components can
access the li
gand and the decreasing number of components can access it until there is only one
component left. To make sure that the maximum capacity of the gel for a certain species does not change
with the model a system of equations has to be fulfilled:

epsilon_vol
ume,i = epsilon_volume,component_j
-

epsilon_volume,component_j
-
1

for all components and all volumes. The capacity in the gel that is not
available for the component is set to 0. The model can be used to approximate the capacity of different
derivatives of

a protein using only the pore sizes and the capacity of one variant of the protein. It has
been used to simulate separation of Bovine Serum Albumin (BSA) from BSA dimers and separation of
monoclonal antibodies (mAb) from aggregates of mAbs.






PREP2010
ABSTRACT/Bowes (12
-
13
-
09)


ABSTRACT #

029


Preference:

Oral


Presenter:

Brian D. Bowes

Department of Chemical Engineering

University of Delaware

Newark, DE 19716

bowes@UDel.Edu



An Automated High
-
throughput Screening Method to Assess Monoclonal Antibody F
it to Common
Purification Process Conditions Using a Single Filter Plate.
Brian D. Bowes
1
, Niels Richings
2
, Joe
Naemura
2
, Thao Nguyen
2
, Ron Gillespie
2
, John Moscariello
2
,
1
Department of Chemical Engineering,
University of Delaware, Newark, DE 19716, USA;
2
Purification Process Development, Amgen, Seattle,
WA 98119, USA


The use of purification platforms for monoclonal antibodies allows for the rapid development of un
-
optimized processes that are suitable for early stage toxicological and clinical trials. For

platform process
development, effort is focused on a small number of resins operated in a narrow window of solution
conditions rather than on understanding entire operating ranges for each unit operation. In considering
the use of high
-
throughput screenin
g to select purification conditions, much of the effort to date has
focused on the optimization of individual unit operations. However, by focusing the information collected
for any given operation to the resins and conditions seen in platform processes, m
ultiple unit operations
can be assessed at once, reducing total screening time and material requirements. In this work, a Tecan
robotic system has been used to create a high
-
throughput screening method to assess the fit of
monoclonal antibodies (mAbs) to o
perating conditions for unit operations typically employed in process
platforms. These unit operations include affinity chromatography steps such as Protein A (ProA), a bind
-
and
-
elute cation exchange (CEX) step, and flow
-
through steps such as anion exchang
e (AEX) and
hydrophobic interaction (HIC) chromatography. A single filter plate is divided into quadrants helping to
make the method adaptable to alternative resin functionalities. Two quadrants are primarily devoted to
determining the binding capacity for

the affinity step and the respective loading and eluting conditions for
CEX. The other two quadrants are used to assess binding of the various species during flow
-
through
operation. The primary data collected from the method are the unadsorbed amounts of

product, high
molecular weight species (HMW), and host cell protein (CHOP) for the chosen combinations of resins and
solution conditions. The high
-
throughput method is shown to adequately determine which unit operations
will be problematic for specific an
tibodies. Lastly, the current screening method is successful in directing
purification process development with the goal of using only two chromatography steps if possible.
Examples will be shown that this method is able to predict which two
-
column proce
ss would be best
implemented to ensure clearance of impurities including viruses.






PREP2010 ABSTRACT/ Cristancho (12
-
1
-
09)


ABSTRACT #

030


Preference:

Poster


Presenter:

M.Sc. Carlos

Andrés Martínez Cristancho

Ph.D Student

Max
-
Planck
-
Institut für

Dynamik komplexer technischer Systeme

Research Group: Physical and

Chemical Foundations of Process Engineering

Prof. Dr.
-
Ing. Andreas

Seidel
-
Morgenstern

Sandtorstraße 1,

D
-
39106,

Magdeburg,
GERMANY

Tel: +49 391 6110

441

crist
ancho@mpi
-
magdeburg.mpg.de




Chromatographic Purification of Single
-
chain Fragment Variable Antibodies.
Carlos Martínez Cristancho
1
,
Andreas Seidel
-
Morgenstern
1,2
,
1
Max
-
Planck
-
Institut for Dynamics of Complex Technical Systems,
Magdeburg, GERMANY;
2
Chair
of Chemical Process Engineering, Otto
-
von
-
Guericke University,
Magdeburg, GERMANY


Recombinant antibodies are the fastest growing class of novel therapeutic proteins
1
. Because of their
small size, these formats are ideal for clinical diagnostics or researc
h purposes by improving tissue
perfusion and specificity. In the frame of the project “From Gene to Product”
2
, a lysozyme specific
histidine
-
tagged single
-
chain fragment variable antibody is expressed in Bacillus megaterium, a gram
-
positive bacterium. Thes
e kinds of bacteria can secret recombinant proteins direct into the growth
medium and in a significant level. The variable parts of the heavy and light antibody chains connected by
a flexible peptide linker make up the smallest fragment variable antibody w
ith a complete antigen
-
binding
site, the so
-
called single
-
chain fragment variable antibody (scFv). Nowadays, the bioseparations using
linear or stepwise gradients are rapidly increasing. The reason is simple; the isocratic mode is typically
not able to iso
late therapeutic proteins from fermentation broths. In this study, we screened
chromatographic resins, pH and salt
-
gradient conditions for the purification of a single
-
chain fragment
variable antibody found in Bacillus megaterium’s cell culture supernatant
. The work to be presented will
also show the pretreatment of the samples prior to chromatography and the method development of a
preparative chromatographic purification process for biomolecules. A successful design has to optimize
many variables, includi
ng the time
-
consuming analytical assays for measuring the yield, productivity, and
purity. The most suitable chromatographic system and conditions to isolate the target component should
be further optimized and following the determination of the adsorption

isotherms. Goal of the study is to
develop a continuous chromatographic process based on step gradients in a 3
-
zone open
-
loop simulated
moving bed chromatography
3

(SMB) or alternatively, gradient separations using two connected 4
-
zone
SMB units
4
. Referenc
es: (1) Schirrmann, T., Laila, A., Dübel, S., Hust, M. Frontiers in Bioscience (2008)
4576. (2) Sonderforschungsbereich 578. Development of biotechnological processes by integrating
genetic and engineering methods. “From Gene to Product”.
http://www.sfb578.tu
-
braunschweig.de/
. (3)
Keßler, L.C., Gueorguieva, L., Rinas, U., Seidel
-
Morgenstern, A. J.Chromatography A 1176 (2007) 69. (4)
Keßler, L.C., Seidel
-
Morgenstern, A. J.Chromatography A 1126 (2006) 323
.



PREP2010 ABSTRACT/Row (11
-
21
-
09)


ABSTRACT #

031

WITHDRAWN


Preference:

Poster

Topic:
Chromatographic Bioseparations


Presenter:

Prof. Kyung Ho Row

Department of Chemical Engineering

Inha University

Incheon,
KOREA

Tel:+82
-
32
-
860
-
7470

rowkho@inha.ac.kr

Submitter: Tian

Minglei
feitiandezhu@hotmail.com




Solid
-
phase Extraction of Liquiritin and Glycyrrhizic Acid from Licorice using Ionic Liquid
-
based Silica.
Minglei Tian,
Kyung Ho Row
,
Department of Chemical Engineering, Inha University, Incheon 402
-
751
,
KOREA


A synthesis of ionic liquid
-
based silica sorbent was developed by a process involving surface chemical
modification of commercial silica. The obtained material was successfully used as a special sorbent in a
solid
-
phase extraction process to separ
ate the liquiritin and glycyrrhizic acid from licorice. Different
washing and elution solvents, such as pure water, methanol, acetonitrile, methanol:water (v/v) and
acetonitrile/water (v/v) were evaluated. Ionic liquid
-
based silica sorbent was compared wit
h traditional C
18

sorbent and it exhibited higher selectivity of the two target compounds. Quantitative analysis was carried
out by using a C
18

column. Good linearity of two compounds were obtained from 5×10
-
4

to 0.2 m
g/mL

(r
2
>0.99) with the relative stand
ard deviations <1.0%. The target compounds in commercial herbal
medicines containing licorice were determined, and the bound rates between the target compounds and
protein were obtained by this sorbent.






PREP2010 ABSTRACT/11
-
24
-
09


ABSTRACT #

032


Preference:

Oral


Presenter:

Emily Belcher Schirmer

Percivia, LLC

1 Hampshire Street 5
th

Floor

Cambridge, MA 02149

617
-
301
-
8837

eschirmer@percivia.com



A Disposable Downstream Process for the Purification of a

Monoclonal Antibody.
Emily Belcher
Schirmer
, Percivia, LLC, 1 Hampshire Street 5
th

Floor, Cambridge, MA 02149, USA


Advances in single
-
use and disposable technologies can enable greater speed, flexibility, and a smaller
footprint for multi
-
product facili
ties, such as a contract manufacturer. In a disposable scheme, traditional
chromatography column operations are replaced with adsorptive membranes or other non
-
traditional
purification methods. Although membrane chromatography offers fast processing times
due to negligible
mass transfer effects, it is often plagued with lower binding capacities. Therefore, the impurity burden
must be minimized at early stages of the process, e.g. clarification, to enable a successful downstream
process. The purification ste
ps implemented in a fully disposable process should meet the requirements
of low cost, easy implementation and operation, and scalability. In this work, a disposable downstream
process was developed for the clarification and purification of a monoclonal an