Liquidations de fin d'année

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Décembre 2011 Issue 91 I NEW sEriE sYsTEC Autoclaves Promo Esco safety Cabinet Promo & NEW Water Purification Elga Purelab Flex NEW Centrifugation Allegra X-30 Pages 24 et 25 Liquidations de fin d'année Prix excePtionneLs !
  • capillary electrophoresis
  • post-translational modification
  • been associated
  • modification post-traductionnelles
  • modifications post-traductionnelles
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  • carbohydrate separation
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Publié le : mardi 27 mars 2012
Lecture(s) : 43
Source : analis.be
Nombre de pages : 28
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Sommaire
Capillary Electrophoresis
06 separation of Fucosylated and non-Fucosylated carbohydrates Associated
with Monoclonal Antibodies using capillary electrophoresis i BEckMAN coULTER
Centrifugation
10 introduction to ultracentrifugation - Part 3 i BEckMAN coULTER
O -CO -pH Detection2 2
14 Development of a shaken scale-Down Model i PRESENS
Water Purifcation
18 Purifed water for mammalian and bacterial cell culture i ELGA
91 Product view
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Décembre 2011 PreSenS I SFrS erlab I asura
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www.analis.be/fashmag 05 electrophorèse capillaire neW 22 Hotte à fux laminaire Action sPéciALe
®bter I ceSI-mS eSco I airstream
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InForS I multitron cell Smeg I Série gW 4090 & gW 3060Eliane Henri : ehi@analis.be
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®elga I Purelab™ fex ohauS I explorerProduction
20 réfrigérateur vertical ProMo 28 Autoclaves neW
Sylvie Mathelot angelantonI I ekofrigolab Série tn SYStec
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20 centrifugeuse Action sPéciALe
hettIch
t irage
2 280 exemplaires
Liquidations de fn d'annéeDiffusion
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Expertise Support Local PartnerneW cesi-Ms
www.beckmancoulter.com/CESI
Un système d’électrophorèse capillaire spécialement conçu pour le
couplage à la spectrométrie de masse (MS)
Le champ d'application de la spectrométrie de masse technologies telles que la LC-MS à phase inversée, sont
va pouvoir être étendu grâce à la combinaison de maintenant bien visibles grâce au CESI 8000.
l’électrophorèse capillaire à haute performance et du
“Le CESI 8000 a été conçu en collaboration avec des
nouveau module ESI de Beckman Coulter. Les débits ultra
chercheurs en spectrométrie de masse qui utilisent
faibles atteints par ce système augmentent la sensibilité
plusieurs applications différentes, et qui cherchaient à
de la spectrométrie de masse et tout en réduisant le
étendre la gamme d’analytes détectés et à augmenter
phénomène de suppression d’ions.
la sensibilité globale” explique Jeff Chapman, director of
Orange County, Calif. – (le 2 juin 2011) Beckman Coulter Discovery Products. “Le résultat est un
Le CESI 8000 High-Performance Separation-ESI Module système de séparation « frontend » qui génère des données
avec la Technologie OptiMS de Beckman Coulter, Inc. complémentaires à HPLC et qui fournit aux chercheurs
comprend le premier atomiseur CESI commercialement d’importantes informations qui n’étaient pas disponibles
disponible en combinaison avec le nouveau système auparavant.
d’électrophorèse capillaire conçu spécialement pour
Le passage aisé de LC-ESI-MS à la CESI-MS permet
la spectrométrie de masse (MS). Ainsi, les chercheurs
l’intégration de CESI dans les processus d’analyse existants.
disposent d’une interface puissante et très sensible entre
Le CESI 8000 est monté sur une paillasse mobile, réglable
l’électrophorèse capillaire (CE) et la MS, et en même
en hauteur pour pouvoir l’ajuster rapidement à la hauteur
temps d’une gamme d’analytes étendue complémentaire
du spectromètre de masse. Des adaptateurs ‘plug-and-
à LC-MS et GS-MS.
spray’ existent déjà pour toutes les grandes marques de
La technologie OptiMS se caractérise par une séparation spectrométrie de masse.
CE à débit extrêmement bas (jusqu’à 10 nl/min) qui est
combiné avec l’ionisation par électronébulisation (ESI) au
cours d’un seul et unique processus dynamique dans
le capillaire même. On peut donc parler de CESI. Pour
accomplir la CESI, l’extrémité du capillaire doit être poreuse
au fux ionique de façon à fermer le circuit électrophorétique
tout en permettant d’entamer le processus ESI dans le
même appareil.
La CESI-MS génère des séparations de peptides avec
des pics élevés (> 320) ce qui permet d’analyser des
échantillons très complexes au niveau protéomique. Les
débits ultrafaibles utilisés en CE, réduisent effectivement la
suppression d’ions et permet de détecter des modifcations
post-traductionnelles (PTMs) comme la phosphorylation et
la sialylation qui typiquement gênent l’ionisation. Grâce à
l’effcacité et la sensibilité des séparations, il est possible
de détecter des peptides qui ne sont présents qu’en
très faibles quantités, à savoir dans des concentrations
picomoléculaires. En plus, la sélectivité de la CE dans
des séparations « frontend », permet la caractérisation
de protéines intactes, de leurs isoformes, de fragments
clivés et de complexes.
Le CESI 8000 avec la Technologie OptiMS de Beckman
Coulter permet également une séparation rapide et
effcace de composants pharmaceutiques, métabolites
pharmaceutiques, acides organiques, acides aminés,
amines à faible poids moléculaire, peptides, acides
nucléiques et nucléosides. Des molécules souvent
pas remarquées ou pas du tout détectables avec les www.analis.be>products>capillary electrophoresis
Info : Luc Van Laer I Tél. 09 243 77 10 I lvl@analis.be Capillary Electrophoresis
separation of Fucosylated and non-Fucosylated
carbohydrates Associated with Monoclonal
Antibodies using capillary electrophoresis
Abstract mab carbohydrate heterogeneity analysis and quantitation is
essential as oligosaccharides linked to their Fc region play an
In order to gain a comprehensive understanding of therapeutic important role in regulation of cell-dependent cytotoxicity (cDc)
1monoclonal antibody (mab) function, it is necessary to critically and antibody-dependent cellular cytotoxicity (aDcc) . Increase
characterize glycosylation associated with them. carbohydrates, in terminal galactose (gal) on mab n-linked oligosaccharides has
2and therefore glycosylation, are known to play an important role been implicated in up-regulation of cDc . therefore, separation
in the structure, function, and clearance of mabs and have been and quantifcation of gal-containing oligosaccharides is benefcial
shown to be responsible for invoking immune responses in to better understand mab function. glycan species varying in
humans. changes in carbohydrate composition or concentration terminal gal content can be readily separated and analyzed using
can signifcantly impact the overall effcacy of therapeutic existing ce technology. glycan sample preparation includes
mabs and can also lead to side effects. Presence of fucose on addition of both charge and fuorescence properties, allowing
monoclonal antibody associated n-linked oligosaccharides is a oligosaccharides to be electrophoretically separated and then
notable glycan modifcation and has been linked to a decrease in detected using laser-induced fuorescence (lIF) technology.
antibody dependent cellular cytotoxicity (aDcc). accurate analysis First, are removed from the asn297 residue
of fucosylated and afucosylated oligosaccharides is therefore of the mab backbone using the n-glycosidase Pngase F. this
critical for a complete understanding of mab microheterogeneity. is followed by derivatization of the fuorophore aminopyrene tri-
capillary electrophoresis (ce) technology has been successfully sulfonic acid (aPtS) via reductive amination at the reducing end
used to separate major Igg n-linked oligosaccharides g0, g1, of the oligosaccharide (Figure 1A). electrophoretic separation can
and g2 structures from one another. the basis for this
separation relies on electrophoresis of
labeled with amino pyrene tri-sulfonic acid (aPtS).
the complexity of glycans associated with many
molecules calls for high resolution separation in order
to assess heterogeneity among carbohydrate isomers
and co-migrating carbohydrate species. Since ce is
already an established technology for automated and
quantitative analysis of n-linked oligosaccharides,
we set out to develop a methodology by which
fucosylated oligosaccharides can be differentiated from
afucosylated species. optimization of chemistry and
ce methods enabled separation of fucosylated and
non-fucosylated carbohydrates from each another.
Introduction
Immunoglobulins or antibodies are soluble serum
glycoproteins involved in passive immunity against
foreign antigens. mono-specifc or monoclonal
antibodies (mab) have been developed as therapeutic
reagents because of their specifcity towards
particular molecular targets associated with disease
manifestation. there exists a high degree of structural
and functional heterogeneity among antibodies, due
in large part to the diversity of associated glycan
populations. glycosylation on therapeutic monoclonal
antibodies is a critical post-translational modifcation
that has been associated with bioactivity, structure, and
pharmacokinetics. a number of different carbohydrate
moieties can potentially associate with mabs, but it
Figure 1. Schematic of glycan analysis sample preparation. A. Glycan cleavage and is generally thought that a core group of bi-antennary
APTS derivatization strategy for of N-linked oligosaccharides. B. Fucosylated and high-mannose structures make up the most
N-linked oligosaccharide.common species.
www.analis.be>products>capillary electrophoresis
6 Biotech 91 Info : Luc Van Laer I Tél. 09 243 77 10 I lvl@analis.be Capillary Electrophoresis
be performed utilizing a polymeric separation matrix consisting of species suggests that it should also be capable of separating
0.4% polyethylene oxide (Peo). beckman coulter has developed fucosylated from afucosylated species.
and commercialized technology (bcoulter p/n 477600)
to help automate and simplify this process. It has been shown Methods and Materials
that the principle for this ce separation of oligosaccharides is
3based on both their mobility and hydrodynamic volume . this all separations were performed using the Pa 800 plus
is illustrated in part by the fact that positional isomers, although Pharmaceutical analysis System confgured with a 488 nm
identical in mass, can be resolved from one another (Figure 2). solid state laser and lIF detection using an emission band-
the presence of a core fucose moiety on mab n-linked pass flter of 520 nm ± 10 nm (beckman coulter Inc). n-cho
oligosaccharides has been associated with a decrease in aDcc capillaries were used for separation of oligosaccharides. all other
4activity and thus reduced effcacy (Figure 1B). It is suggested assay conditions were as described in the standard operating
that therapeutic mabs produced in various cell lines such as the procedure for the carbohydrate labeling and analysis assay
commonly used chinese hamster ovary (cho) cells contain kit, (beckman coulter p/n 477600) with the exception that the
5glycans that are >90% fucosylated . because of this, separation carbohydrate separation buffer was substituted with a new
of these species is necessary for accurate analysis. Due to a separation buffer formulation where indicated. Final concentration
size difference as small as 16 daltons between fucosylated and for oligosaccharide samples was 1.25 mm. glycan standards
afucosylated glycans, as well as the presence of numerous for fucosylated and afucosylated species of g0, g1, g1’, and
positional isomers, separation has proven to be diffcult. current g2 were purchased from glyko ProZyme, Inc. (hayward, ca).
methods have been incapable of resolving all of the major co- the therapeutic mab was obtained from genentech, Inc. (S San
migrating glycan species from one another. the success for ce Francisco, ca).
technology to resolve terminal gal differences on oligosaccharide
Figure 2. Separation of G0,
G1, and G2 glycan species.
Representative data (top trace)
shows separation of N-linked
oligosaccharides G0, G1, G1’,
and G2 using the Carbohydrate
Labeling & Analysis Assay Kit
(Beckman Coulter p/n 477600).
G1 positional isomers are
resolved from one another
illustrating the mobility- and
hydrodynamic volume- based
separation. The bottom trace
shows separation of a glucose
ladder standard. The ‘G’
designation for the glucose ds refers to the
number of glucose subunits
making up that standard.
Figure 3. G0, G1, and G2
oligosaccharides. G0, G1, and G2
oligosaccharides are commonly
associated with monoclonal
antibodies. The ‘G’ designation refers
to the number of galactose subunits
occupying the bi-antennary termini of
the oligosaccharides. Note G1 can
exist as either one of two positional
isomers differing in only the location
of the terminal galactose.
www.analis.be>products>capillary electrophoresis
Info : Luc Van Laer I Tél. 09 243 77 10 I lvl@analis.be Biotech 91 7Capillary Electrophoresis
Experimental details for this work were as follows: Conclusions
- carbohydrate separation gels used high resolution capillary electrophoresis separations based on
- carbohydrate assay gel (contains polyethylene oxide (Peo)) mobility and hydrodynamic volume have been developed for
buffer or, quantitative analysis of glycans. using published protocols and
commercially available reagents, we have shown this technology - new separation gel buffer was 1:1 mixture of:
to be capable of separating oligosaccharides differing in - carbohydrate separation gel buffer (Peo) – bec p/n 477623
terminal galactose. We also showed that by combining the - dsDna1000 separation gel buffer (lP a) – bec p/n 477628
standard Peo separation gel buffer with a lP a gel buffer, we
- capillary length: total length = 60.2 cm, length to were able to separate fucosylated from afucosylated n-linked
detector = 50 cm
oligosaccharides. this work suggests that ce can be used to
- capillary diameter: 50 mm I.D. successfully separate and quantify n-linked oligosaccharide
populations associated with mabs. additional experimentation - Injection conditions: 0.5 psi for 10 sec
will focus on development of these methods.- Separation voltage: 30 kv
- Field Strength: 500 volts/cm
- capillary cartridge temperature: 20° c
- Sample storage temperature: 10° c
Figure 4. G0+fucose (G0F) &
G1’+fucose (G1’F) co-migrate
with G1 & G2 respectively. Spiking
experiments were performed and
illustrated co-migration of G0F with
G1 oligosaccharide species as
well as co-migration of G1’+fucose
(G1’F) with G2. This separation
was performed using the standard
separation buffer and conditions
described in the Carbohydrate
Labeling & Analysis Assay (Beckman
Coulter p/n 477600).
Figure 5. Optimization of the
carbohydrate separation buffer
allows for resolution between co-
migrating oligosaccharide pairs.
Using standard sample preparation
protocols, oligosaccharide standards
were APTS labeled and separated
by CE. Resolution of co-migrating
fucosylated and afucosylated
N-linked oligosaccharide standards
was facilitated by combining existing
Beckman Coulter separation buffers.
Separation buffer consisted of a 1:1
mixture of Carbohydrate Separation
Buffer (Beckman Coulter p/n
477623) containing 0.4% PEO and
dsDNA 1000 Gel Buffer (Beckman
Coulter p/n 477628) containing
a fnal concentration of 6% linear
polyacrylamide (LPA). Co-migrating
species G0F and G1 (red labels) as
well as G1’F and G2 (blue labels)
were separated from one another.
www.analis.be>products>capillary electrophoresis
8 Biotech 91 Info : Luc Van Laer I Tél. 09 243 77 10 I lvl@analis.be Capillary Electrophoresis
Results and Discussion shown in Figure 3. Spiking experiments using oligosaccharide
standards illustrated that individual separated peaks may contain
the goal of this study was to achieve separation of the major multiple glycan species. this was demonstrated by co-migration
glycan species associated with monoclonal antibodies. this of g0+fucose (g0+F) with g1, and co-migration of g1’+fucose population includes positional isomers as well as (g1’+F) with g2 (Figure 4). modifcation of separation parameters
fucosylated and afucosylated oligosaccharides. We set out to such as capillary length, separation voltage, and temperature did
characterize the separation limitations for an existing separation not offer improved resolution (data not shown). by developing
chemistry, the beckman coulter carbohydrate labeling and a new separation buffer formulation, we were better able to
analysis assay kit. employing standard kit protocols for resolve these co-migrating species (Figure 5). In order to test
instrument confguration, sample preparation, and separation this separation method on a real molecule, we obtained a
conditions, we easily attained baseline resolution between g0, therapeutic mab and analyzed glycans associated with it (Figure 6).
g1 positional isomers (g1 and g1’), and g2 oligosaccharide Spiking with oligosaccharide standards to help identify glycan
species (Figure 2). a systematic approach was devised in which species, we showed good resolution between many of the major
standards were spiked into samples to help identify additional oligosaccharides including g1’+F and g2, which were previously
peaks in this separation and also to better defne co-migration of diffcult to separate by ce.
glycans that may be occurring. the g0, g1 and g2 species are
Figure 6. Separation of oligosaccharides
associated with a recombinant therapeutic
MAb. Oligosaccharides were cleaved from
a therapeutic MAb, APTS labeled, and
separated by CE using the new buffer
formulation. A number of oligosaccharide
species were resolved from one another
(A). In order to identify and help illustrate
resolution between co-migrating glycan
species, we spiked the MAb sample with
standards. Relative to the oligosaccharide ds, we were able to quantifably
identify G0, G0F, G1F, G1’F, and G2F. G2
standard was also spiked into the mixture
to indicate the location in the separation at
which this oligosaccharide species would
reside. Further experimentation is being
performed to identify the additional species
present in the electropherogram. Separation
conditions were the same except that
injection for the MAb alone was 0.5 psi and
that for the MAb + glycan standards was
1.5 psi.
Sushma Rampal, Oscar Salas, and Mark Lies
References
1. raju, tS. glycobiology (2008) 20: 471-478. I 2. hodoniczky et al. biotechnol Prog (2005) 21: 1644-1652. I 3. guttman et al. electrophoresis (1996) 17: 412-417.
4. Shields et al. J biol chem (2002) 277: 26733-26740. I 5. raju et al. glycobiology (2000) 10: 477-486
www.analis.be>products>capillary electrophoresis
Info : Luc Van Laer I Tél. 09 243 77 10 I lvl@analis.be Biotech 91 9Centrifugation
Beckman coulter
introduction to ultracentrifugation - Part 3
1.2 Zonal rotorsAfter the article on the different gra-
dient materials, types of gradients Zonal rotors are flled and emptied directly
during centrifugation. In the case of gradient and their preparation published
centrifugation, this replaces the normal function in the last issue of FLAsHmag 90,
of a swing-out rotor so that, although a zonal in this part of the introduction, we
rotor looks like a fxed angle rotor, the gradient would like to present the rotors for
and sample loading process causes a layering gradient centrifugation as well as their
effect similar to a swing-out rotor for gradient Fig. 3 cross-section of a fxed angle rotoradvantages and disadvantages. centrifugation.
1.3 Fixed angle rotors
In the fxed angle rotors, the centrifuge tubes
are located at specifc angles to the rotor axis
(fg. 3). the angles are different from rotor to
rotor, in ultracentrifuge rotors the angles mostly
Fig. 4 cross-section of a vertical rotor range between 20° and 35°. compared to
swing-out rotors, the fxed angle rotors have
a smaller separation distance, which results in
a shorter separation time.
Fig. 1 cross-section of the swing-out rotor
Rotors for gradient centrifugation 1.4 Vertical rotors
having been developed for minimum basically, all types of rotors may conceivably
separation time, the separation distance, and be used, and in fact gradients may be
Fig. 5 cross-section of a near vertical rotor in consequence the time, is as centrifuged in fxed angle, vertical, near vertical
small as possible on account of their particular and swing-out rotors. and if their capacity is
geometry. the pockets for the centrifuge not suffcient for the sample volume, scaling
tubes are fxed vertically, and hence are at a up to zonal and continuous fow rotors is
right angle with respect to the centrifugal feld possible. In both types of these rotors, the
during centrifugation (Fig. 4).sample may be added and withdrawn during
centrifugation. Depending on the experiment,
the rotors should be regarded as swing-out
1.5 Near vertical rotors
rotors or as fxed angle rotors. What matters
near vertical rotors are actually also fxed is that the quality of the sample purifcation by
angle rotors; however, the angle with respect means of gradient centrifugation depends on
to the rotor axis is clearly smaller, ranging the used rotor type.
between 7° and 9°. hence, these rotors are
closer to the vertical rotors than to the fxed 1) Available rotor types
angle rotors, where their name is derived
1.1 swing-out rotors from. the near vertical rotors were developed
by beckman coulter in order to combine In swing-out rotors, the centrifuge tubes are
the advantages of fxed angle rotors with located in buckets, which whilst the rotor is at
the of vertical rotors. they are rest, i.e. prior to centrifugation hang vertically
exclusively available from beckman coulter. and swing out during centrifugation and
the angles of the centrifuge containers are are then at a 90° angle with respect to the
designed such that a pellet forms as in a fxed rotor axis (Fig. 1). the centrifuge tubes are
angle rotor and the separation distance and in thus always exactly aligned in the respective
consequence the time is similar to accelerating feld (i.e. in the gravitational feld
that of vertical rotors.during standstill, and in the centrifugal feld centrifugation. the forces always apply
Fig. 2 Zonal rotor Ti 15in the direction of the tube axis.
www.analis.be>products>ultracentrifuges
10 Biotech 91 Info : Vincent Jadin I Tél. 081 25 50 50 I vjn@analis.be

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