-
Journal of Chromatography A, 1376 (2015) 7483
Contents lists available at ScienceDirect
Journal of Chromatography A
jo ur nal ho me pag e: www.elsev ier .com/ locate /chroma
Fabricating electrospun cellulose nanobre adsorion-exc
Stewart . Bra Department o C1H b Innovations T , ScienOxford,
Didcot OX11 0QX, UKc School of Science and Technology, Nottingham
Trent University, Nottingham, NG1 4BU, UK
a r t i c l e i n f o
Article history:Received 24 JuReceived in reAccepted 4
DeAvailable onlin
Keywords:ElectrospinninConvective
maDiethylaminoeCarboxylateTEMPO-media
a b s t r a c t
Protein separation is an integral step in biopharmaceutical
manufacture with diffusion-limited packed
1. Introdu
The conprescriptionestimated t
CorresponCollege LondoTel.: +44 0 20
E-mail add
http://dx.doi.o0021-9673/ ly 2014vised form 2 December
2014cember 2014e 12 December 2014
gss transferthyl
ted oxidation
bed chromatography remaining the default choice for industry.
Rapid bind-elute separation using con-vective mass transfer media
offers advantages in productivity by operating at high owrates.
Electrospunnanobre adsorbents are a non-woven bre matrix of high
surface area and porosity previously inves-tigated as a
bioseparation medium. The effects of compression and bed layers,
and subsequent heattreatment after electrospinning cellulose
acetate nanobres were investigated using diethylaminoethyl(DEAE) or
carboxylate (COO) functionalisations. Transbed pressures were
measured and compared bycompression load, COO adsorbents were 30%,
70% and 90% higher than DEAE for compressions 1, 5 and10 MPa,
respectively, which was attributed to the swelling effect of
hydrophilic COO groups. Dynamicbinding capacities (DBCs) at 10%
breakthrough were measured between 2000 and 12,000 CV/h (2 s and0.3
s residence times) under normal binding conditions, and DBCs
increased with reactant concentrationfrom 4 to 12 mg BSA/mL for
DEAE and from 10 to 21 mg lysozyme/mL for COO adsorbents.
Comparingcapacities of compression loads applied after
electrospinning showed that the lowest load tested, 1 MPa,yielded
the highest DBCs for DEAE and COO adsorbents at 20 mg BSA/mL and 27
mg lysozyme/mL, respec-tively. At 1 MPa, DBCs were the highest for
the lowest owrate tested but stabilised for owrates above2000 CV/h.
For compression loads of 5 MPa and 10 MPa, adsorbents recorded
lower DBCs than 1 MPa asa result of nanobre packing and reduced
surface area. Increasing the number of bed layers from 4 to
12showed decreasing DBCs for both adsorbents. Tensile strengths
were recorded to indicate the mechanicalrobustness of the adsorbent
and be related to packing the nanobre adsorbents in large scale
congu-rations such as pleated cartridges. Compared with an
uncompressed adsorbent, compressions of 1, 5and 10 MPa showed
increases of 30%, 110% and 110%, respectively, for both
functionalisations. The datapresented show that capacity and
mechanical strength can be balanced through compression after
elec-trospinning and is particular to different functionalisations.
This trade-off is critical to the development ofnanobre adsorbents
into different packing congurations for application and scale up in
bioseparation.
2014 The Authors. Published by Elsevier B.V. This is an open
access article under the CC BY
license(http://creativecommons.org/licenses/by/3.0/).
ction
tribution of biotechnology products to the global and
over-the-counter pharmaceutical markets wereo be worth $118 billion
in 2011 with increased focus
ding author at: Department of Biochemical Engineering,
Universityn, Bernard Katz Building, Gordon Street, London WC1H 0AH,
UK.7679 9580; fax: +44 0 20 7209 0703.ress: [email protected]
(D.G. Bracewell).
in the therapy areas of oncology, anti-diabetes and vaccines
[1].Some individual products are reaching annual sales of over
$1billion [2]. As the market moves towards developing more com-plex
biomolecules such as fusion proteins and antibody
fragments,purication stages in downstream processing are becoming
moreexpensive. The advancement of cell line engineering in
upstreamprocessing, including transfection methods and media
develop-ment, in upstream processing have realised increased
product titresover the past two decades [3]. However, downstream
processinghas yet to achieve a dramatic improvement in process
ef-ciency partly due to limitations in widely used packed-bed
resins
rg/10.1016/j.chroma.2014.12.0102014 The Authors. Published by
Elsevier B.V. This is an open access article under the CC BY
license (http://creativecommons.org/licenses/by/3.0/).hange
chromatography
R. Dodsa,b, Oliver Hardicka, Bob Stevensc, Daniel Gf Biochemical
Engineering, University College London, Bernard Katz Building,
London Wechnology Access Centre Micro and Nanotechnology,
Rutherford Appleton Laboratorybents for
acewell a,
0AH, UKce and Technology Facilities Council, Harwell
-
S.R. Dods et al. / J. Chromatogr. A 1376 (2015) 7483 75
including diffusive mass transfer, achievable ow rates and
scale-up volumes. Protein bioseparation media using convective
masstransfer such as porous membranes and monoliths have
receivedincreased attention because they avoid this diffusion
limitation andhave a higher capture efciency and reduced buffer use
to improveoverall productivity [4]. In the last 30 years, rigid
porous monolithshave also been introduced and developed. The single
solid contin-uous matrix has no interstitial voids and can also
vastly improveproductivity by operating at much higher owrates than
packed-bed chromatography [5]. Current advantages in industry have
beenrealised in the polishing stage of monoclonal antibody
purica-tion using owthrough mode where a membrane column
bindsimpurities and allows the target to pass through [6].
Nanobre electrospinning involves passing a viscous
polymersolution through a microneedle charged at a high voltage
(>5 kV)to deposit a continuous bre strand to a grounded
collector andform a non-woven mat with a bre diameter of less than
1 m[7]. Electrospun nanobres have been investigated for a
mul-titude of applications including tissue engineering [8],
catalysisand sensors [9,10], ltration [11] and composites [12].
Celluloseis a commonly used material in membrane chromatography
andltration for being chemically resistant, cheap and has good
non-specic binding properties [4]. However, cellulose raises
manychallenges in electrospinning because it is difcult to dissolve
andthe solvent systems required can lead to non-uniform
nanobredeposition lulose derivby regenerabre depos(viscosity),
ditions havacetate nancal strengthFig. 1 showdifferent mresin and
anadsorbent. porosity wi
Chemicahydroxyl grhave been bioseparatimers
includlaminoethyfabricated b
ethyl chloride hydrochloride (DAECH) to show improved
sepa-ration productivity compared with porous membranes
[19,20].Alcohol groups on cellulose have been controllably oxidised
tocarboxylate (COO) groups using
(2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) as a catalyst and
sodium hypochlorite as theoxidant [21,22]. The main application of
TEMPO-mediated oxida-tion is in the preparation of nanocellulose
from wood pulp wherethe ionic repulsion of COO groups helps force
cellulose bres apartduring processing, reducing the mechanical
energy required [23].The use of TEMPO-mediated oxidised electrospun
cellulose nano-bre has been used before to bind metal ions [22] and
viruses[24]. The physical and chemical methods applied in
fabricatingelectrospun cellulose nanobre adsorbents affect
bioseparationperformance and controlling parameters is important to
fabricatinga reproducible material. Compressing nanobre sheets
combinedwith annealing via heat treatment is used to further
improvemechanical properties than heat treatment alone. A robust
nano-bre adsorbent is essential in scaled up packed bed
congurationssuch as pleated cartridges, as seen in membrane
chromatogra-phy. However, chemical modications applied to nanobres
mayadversely affect morphology and structure.
2. Materials and methods
2.1. Fabricating cellulose nanobre adsorbents
shor cons
i.d. withity se
(Mrn watio as sp(200 age sneed00 m2, whus stquarn tw
die.lic p
Fig. 1. Scanni nd S cand heat treat ked-bediameters and[13]. As
such, electrospinning readily dissolvable cel-atives such as
cellulose acetate are preferred followedtion to cellulose via
hydroxide treatment. For uniformition of cellulose acetate,
controlling polymer solutionow rate and voltage as well as
environmental con-e been shown to be critical [14]. Annealing
celluloseobres with heat is a common step to improve mechani-
by creating spot welds at bre strand overlap points.s scanning
electron microscopy (SEM) images of theorphologies for a cast
porous membrane, packed-bed
annealed electrospun regenerated cellulose nanobreA nanobre
adsorbent balances a high surface area andth the benets of
convective mass transfer.l modications of chromatographic media
usingoups on the support for application in bioseparationresearched
[15]. Electrospun nanobre adsorbents inon have been reported for
cellulose [16] and other poly-ing polysulfone [17] and
polyacrylonitrile [18]. Diethy-l (DEAE) cellulose electrospun
nanobres have beeny Williamson ether synthesis using
2-(diethylamino)
To mat of0.5 mmin linehumidacetatesolutioat a rtion wdrum tion
stof the sheet 630 g/mprevio[19]. Sbetweeas thehydrau
ng electron microscopy images comparing protein purication
media. (a) Sartobied regenerated cellulose nanobre adsorbent. (c)
Fractogel EMD TMAE HiCap pac
approximately 0.1 m pore diameter.ten the electrospinning time
and produce nanobreistent bed height, four microneedles (100 mm
length;) were used and the collector was moved side-to-side
the needle array. The operating voltage was 30 kV, thet to 70%
and temperature to 25 C. A 20 wt.% cellulose
= 29,000, 40% acetyl groups, Sigma-Aldrich, Dorset, UK)as
prepared in acetone:DMF:ethanol (SigmaAldrich)of 2:2:1 as
previously described [14,19]. The solu-un at 2.5 mL/h for 10 h. The
collector was a rotatingmm dia.; 300 mm length) set at 60 rpm on a
transla-et at 300 mm x-axis displacement (150 mm either sidele
array centre) at a rate of ve loops per minute. Am 180 mm was
produced equating to approximatelyich was comparable to the nanobre
mat used in our
udy but a reduction in spinning time from 36 h to 10 hes (80 mm
80 mm) were cut, layered and placed ino 100 mm 100 mm square
aluminium blocks to act
Compression was performed for 2 min in a manualress (Specac,
Kent, UK) under different loads of 1000,
ellulose membrane (Sartorius Stedim, Epsom, UK). (b) Compressedd
resin (EMD Millipore, Darmstadt, Germany) with 4090 m bead
-
76 S.R. Dods et al. / J. Chromatogr. A 1376 (2015) 7483
5000 and 10,000 kg as indicated on the gauge, corresponding
to0.98, 4.9 and 9.8 MPa, respectively, for brevity, we used
roundedup values; 1, 5 and 10 MPa, respectively. To study the
effects of themain reactant concentration, 8 layers were compressed
at a load of5 MPa. To scompressedcompressedin a preheafor 30 min.
by deacetylovernight. Tdeacetylatio
2.2. DEAE m
Cellulosform DEAE100 mL deioof DAECH atdamaging trepeat
reaccellulose (2was stirred(90 C) 0.5 Mdissolve anwas rinsed
2.3. TEMPO
A COO-cmodied frous mixturSigma-AldrNaOH. The nused to dropfor
the thrsodium hypitored to enof the C6 hwas 10 minEthanol (10and
stirred pure waterchlorite (Siacid) was p[26].
2.4. Morph
SEM imaWorld BV, Eof 10 keV. ric softwarFourier traATR) was ua
Thermo Sough, UK). 4000500 cmeasured wstrength m10 MPa, weinto a
tensohighest reco
2.5. Equilibrium adsorption capacities
To assess the nanobre equilibrium binding capacity the matswere
cut into 25-mm discs. Discs were incubated in 0.02.0 mg/mL
protV-63ter bAE c
10 mlyso. Thndinbres . Thr
cons (CaxKdCein bearis
bestightaki, perimlculae adroteients
BCs a
dynasicent ausly ution
anddsorpell eq
betw of 2O adessedent amg/m
DEA wit0 cmffer
The equid voteine loae in m anes wAE a, 0.1sorbL, 4 lessels.
Toeaseordelter htudy the effects of physical modication, 8 layers
were at loads of 1, 5 and 10 MPa and 4, 8 and 12 layers were at 5
MPa. The nanobre sheet was immediately placedted oven (NR30F,
Carbolite, Shefeld, UK) set at 213 CCellulose acetate mats were
regenerated to celluloseation using 0.1 M sodium hydroxide in 2:1
H2O:EtOHhe addition of ethanol was essential to ensure
completen.
odication
e adsorbents can be reacted directly with DAECH to ligands via
alkylation [19,20]. A reaction solution ofnised water was employed
with varying concentrations
50 and 200 mmol/g cellulose stirred at 250 rpm to avoidhe
nanobre mat. To improve DEAE functionalisation, ation was performed
using 200 mmol DAECH per gram 200 mmol) as previously reported
[25]. The reaction
for 15 min at 250 rpm. Then the mat was treated in hot NaOH
solution for 10 min to complete the reaction and
y unwanted reactants. The DEAE-cellulose adsorbentin copious
amounts of water.
-mediated oxidation
ellulose adsorbent was produced following a procedureom that
previously reported [21]. A 100 mL aque-e of TEMPO (0.002 g;
Sigma-Aldrich) and NaBr (0.02 g;ich) was adjusted to a pH of 10.5
using aqueous 0.1 Manobre mat was stirred for 5 min. A syringe pump
waswise add sodium hypochlorite (NaClO; Sigma-Aldrich)ee
concentrations investigated; 5, 10 and 20 mmolochlorite (NaClO) per
gram cellulose. The pH was mon-sure pH remained above 10.5 to
encourage oxidationydroxyl on the cellulose. The time taken to add
NaClO
and the mixture was allowed to stir for a further 5 min. mL,
Sigma-Aldrich) was added to quench the reactionfor 10 min. The mat
was washed thoroughly with ultra-. To oxidise any remaining
aldehyde groups, sodiumgma-Aldrich) treatment (0.45 g in 45 mL in 1
M aceticerformed for 48 h in the dark to as previously
described
ological, chemical and tensile strength analyses
ging was performed using a Phenom G2 Pro (Phenom-indhoven, The
Netherlands) at an accelerating voltageImages were captured and
analysed with Firbomet-e (Phenom-World BV) to estimate bre
diameter.nsform infra-red attenuated total reectance (FTIR-sed to
characterise the chemical group changes oncientic Nicolet iS10
FT-IR Spectrometer (Loughbor-Spectra were recorded from dry samples
in the rangem1 by an accumulation of 50 scans. A background wasith
10 scans prior to each sampling. For ultimate tensile
easurements, compressed adsorbent samples, 1, 5 andre cut into
15 mm 10 mm (L W) strips and placedmeter. The strips were stretched
at 1 mm/min and therded force before breaking was used.
model(Jasco ing, afthe DEtein instudy, pH 5.5tive
binanotestingproteintrationQ = Qmof protThe linline ofbed
heKawasThe exwas caand thered padsorb
2.6. D
TheAKTA Bsurempreviodistribbuffersrium awas wvariedtrationand
COcompradsorband 5 for theformedat a 61tion bubuffer.lowingthe
vothe promust bvolum0.014 cvolumFor DE10 MPaCOO ad0.20
mnon-prcontroto incrthe recing a ein solutions and UV absorbance
readings at 280 nm0, Essex, UK) were taken at each step of before
bind-inding (16 h), wash (1 h) and elution (1 h). For
testingellulose nanobre, BSA was used as the model pro-M Tris
buffer at pH 8.0. For the COO-cellulose bindingzyme was used in 20
mM sodium acetate buffer ate elution buffers used were the same as
the respec-g buffer and containing 0.5 M NaCl. After elution,
thewere regenerated in 0.1 M aqueous NaOH for repeatee tests were
performed and the adsorbed equilibriumcentrations (Q) and liquid
phase equilibrium concen-) were averaged. The Langmuir adsorption
isotherm/Kd + C was used, where Qmax is the maximum capacityound,
and Kd is the equilibrium dissociation constant.ed form of Langmuir
isotherm was plotted and from a
t the Qmax and Kd values were estimated. The wet was measured
with a digital micrometer (Mitutoyo,Japan; 0.001 mm resolution) to
calculate the volume.
ent was performed three times. Elution performanceted as a ratio
of the protein concentration after elutionsorbed equilibrium
protein concentration. The recov-n concentration was 75% for DEAE
and 90% for COO.
nd transbed pressures
amic binding capacity (DBC) was measured using an (GE
Healthcare, Uppsala, Sweden) system with UV mea-t 280 nm. A
custom-made 25-mm PEEK lter holder wasdesigned using frit spacers
to ensure full radial ow
across an adsorbent at very high owrates [19]. The model
proteins were the same as used in the equilib-tion study. The
nanobre adsorbent in the lter holderuilibrated prior to binding.
The binding owrates wereeen 10 and 610 cm/h. BSA or lysozyme at a
concen-
mg/mL in a 2-mL sample loop was injected for DEAEsorbents
testing in most cases. For 8-layer adsorbents
at 1 MPa all of the protein injected was bound to thend the
protein concentration was increased to 3 mg/mLL of BSA and lysozyme
to provide a maximum DBCE and COO adsorbents, respectively. Elution
was per-h a 30% mix of 1 M NaCl in respective binding buffer/h and
the adsorbent was further cleaned with elu-at 610 cm/h, followed by
re-equilibration with bindingDBC was calculated at 10% breakthrough
using the fol-ation DBC10% = ((V10% V0) + CLoad)/VBed where V0
islume of the entire system, CLoad is the concentration of
solution loaded, and V10% is the volume of sample thatded before
achieving 10% breakthrough. VBed is the bedmillilitre measured from
a bed height range betweend 0.07 cm with a digital micrometer
(Mitutoyo). Bed
ere taken as an average of the three adsorbents
tested.dsorbents: 1 MPa, 0.35 mL; 5 MPa (8 layers), 0.16 mL;6 mL; 4
layers, 0.07 mL; and 12 layers, 0.21 mL. Forents: 1 MPa, 0.34 mL; 5
MPa (8 layers), 0.19 mL; 10 MPa,ayers, 0.09 mL and 12 layers, 0.21
mL. Blank tests usingd unmodied cellulose adsorbents were performed
as
measure transbed pressure, the AKTA was programmed in owrate in
steps up to 50 mL/min and presented asd back pressure minus the
system back pressure includ-older containing no adsorbent.
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S.R. Dods et al. / J. Chromatogr. A 1376 (2015) 7483 77
3. Results and discussion
3.1. Surface morphology
SEM sergies and brepresentatricate DEAEmaterial prdle and
hadopen with (Fig. 2a). Thand the samhere at an awere compduce
regencompact mdiameters rc(ii) show by repeat tr20 mmol NDEAE
adsobre strandbeen repories [19,20]. above protmatrix uponbents
also sin the unmooxidation hpare nanocpolymer chwood pulp assists
in degrade wothe DEAE angroups on tsion forcingnoticeable. nanobre
mwas no lonDAECH for considered in the morpcation.
3.2. FTIR-AT
FTIR-ATRgroups on ththe differenFig. 3. Celluclear from t1740
cm1,(OC O); 12the 33003showed no low concenthe cellulos[19]. Fig.
31731 cm1,salt (COONaC O peak hThe applicawith detect
3.3. Tensile strength
Previous investigations into non-compressed nanobre adsor-bents
suffered damage during chemical modication from mixing
us remogg anful ts wratedand % inessed
imping pnd 1on e
diffe streere tand C
proion hegradpulscatiod thampleutescked
ansb
nsbeadsool D
Fig. g comte wiessioessurositon-cdsorEAE b). C
to ethyg effangey theous
shee beessurecorice asent obee of ed vessuhad c
stre capves as an important tool for investigating morpholo-re
diameters of electrospun nanobres. Fig. 2 showsive SEM images and
modication steps taken to fab-
and COO adsorbents. The cellulose acetate startingior to any
compression or baking was fragile to han-
a cotton-wool like texture. The morphology appearedlarge black
spaces between the straight bre strandse electrospinning conditions
were previously reportede small range in nanobre diameters was
measured
verage of 0.5 m [14]. Eight layers of cellulose acetateressed at
5 MPa, oven-baked and deacetylated to pro-erated cellulose. The
general appearance was a moreatrix with strands retaining a linear
appearance andemained in the same range (Fig. 2b). Figs. 2c(i)
andbre matrices following chemical modications to DEAEeatment of
DAECH (2 200 mmol) and COO groups ataClO concentration per gram
adsorbent, respectively.rbents showed a slightly distorted
appearance withs losing some of their linear character, which has
notted for singly treated DEAE nanobres in other stud-More than two
repeats of DAECH treatment using theocol led to a complete
degradation of the nanobre
drying, becoming a hard opaque material. COO adsor-how a loss of
linear character and distortion not seendied regenerated cellulose
matrix. TEMPO-mediatedas use in the nanobrillation of wood pulp to
pre-ellulose particles and brils. By charging the celluloseains
with COO groups the process of homogenisingconsumes less energy
because ionic repulsion forcesbril separation [23]. The ionic
repulsion is used tood brils into nanobrils 34 nm in diameter [27].
Ind COO nanobre adsorbents here, the effect of charged
he nanobre strand in solution may lead to ionic repul- nanorils
apart that make up the strand becomingUpon drying for SEM analysis
the appearance of theatrix was only slightly distorted because the
surface
ger charged. The chemical conditions of 2 200 mmolDEAE and 20
mmol NaClO for COO adsorbents werethe highest possible before any
considerable changehology rendered the adsorbent unsuitable for
appli-
R
was employed to investigate the changes in chemicale surface of
cellulose nanobre adsorbents throughoutt modication and
representative spectra are shown inlose acetate deacetylation to
regenerated cellulose washe replacement of acetate peaks (ester
carbonyl (C O);
carbon-methyl (CCH3); 1365 cm1 and ester linkage21 cm1) by a
broad and larger alcohol (OH) peak in500 cm1 region. DEAE
modication (2 200 mmol)new peaks because the weak stretching and
relativelytration of the tertiary amine bonds were masked bye
peaks, regardless how high a modication is usedb shows that
COO-cellulose created a new peak at
corresponding to the carbonyl group of the carboxylate). As the
amount of oxidant NaClO was increased, theeight increased,
indicating an increase in COO groups.tion of FTIR-ATR was
convenient to investigate groupsable peaks.
and thand hospinninbe useSampleregeneDEAE and 30compr10
MParecord130% adeviatitisticaltensileples wDEAE ing anyoxidatbre
dionic remodiimpliethis sacontriband pa
3.4. Tr
Trausing 200 mmbents. varyinowracomprbed prand pothan nCOO athan
D(Fig. 5shownboxymswellinThe chsised b
Poras atincreasbed pr5 MPa ing twadsorbof nanincreassmall bbed
prwhich tensilereducequired a less vigorous approach, hindering the
amounteneity of functionalisation. Compressing after electro-d
followed by heat treatment was therefore found too reduce failures
and produce consistent adsorbents.ere compared with non-compressed
and heat treated
cellulose indicated as the No Press sample (Fig. 4).COO
adsorbents compressed at 1 MPa showed a 40%crease in tensile
strengths compared with the non-
sample, respectively. Increasing compression to 5 androved
tensile strength over non-compressed further,ercentage differences
of 85% and 105% for DEAE and20% for COO, respectively. However, the
large standardrrors for 5 and 10 MPa compressions suggest no
sta-rence between them and may indicate a maximum ofngth achieved
for these cellulose adsorbents. The sam-ested when dry and the weak
ion-exchange groups ofOO would be in their neutral form and not
exhibit-
nounced effect from ionic repulsion. TEMPO-mediatedas been used
in nanocellulose production, assistingation during mechanical
processing in solution throughion [27]. The changes in morphology
between chemicalns were negligible and similarities in tensile
strengthst chemical modication has little or no effect, at least
in
size. Improving the mechanical strength of adsorbents to
creating a robust material capable of being handled
into large scale pleated or spiral-wound congurations.
ed pressures of varying bed layers and compressions
d pressures were recorded for increasing owratesrbents prepared
at chemical modications of 2AECH for DEAE and 20 mmol NaClO for COO
adsor-
5a shows similarly increasing transbed pressures forpressions of
8-layer DEAE adsorbents with increasing
th 10 MPa showing the highest of the three. Increasingn during
fabrication was expected to increase trans-res because the nanobre
matrix was more packedy was reduced. DEAE transbed pressures were
higherompressed DEAE adsorbents previously reported [19].bents show
considerably higher transbed pressuresand differences between
increasing compression loadsOO groups are hydrophilic and have been
previouslycause a higher back pressure when comparing car-l - with
DEAE modied cellulose beads, suggesting thisect contributes to
increases in transbed pressure [28].
in matrix packing was clear in COO adsorbents empha- hydrophilic
nature of COO groups.membranes, like nanobre adsorbents, are
producedets and designing a media with multiple layers tod height
is one method to increase bed volume. Trans-res of 4-, 8- and
12-layer adsorbents compressed atded noticeable differences with
COO adsorbents show-
high pressures than DEAE (Fig. 5c and d). The 12-layerbed volume
was similar to 8-layer, but the packingr matrix much higher, which
was evidenced in thetransbed pressure. The 4-layer adsorbents were
of sucholumes that hardly any pressure was recorded. Trans-res
provided an insight to the packing of nanobres,learly increased
with increasing compression from thength results. However, higher
transbed pressures mayacity because of the channelling effect seen
in porous
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78 S.R. Dods et al. / J. Chromatogr. A 1376 (2015) 7483
Fig. 2. Fabricamodication. Carboxylate (C
membranesarea of an a
3.5. Equilibmodication
Reactanusing 8-laytion of electrospun cellulose nanobre
adsorbents and representative scanning electron(b) Regenerated
cellulose adsorbent after 5 MPa compression, heat treatment and
deaceOO) cellulose adsorbent.
where the proteins are not accessing all the
surfacedsorbent.
rium absorption capacities of varying chemicals
t concentrations were varied to investigate capacitieser
adsorbents compressed at 5 MPa (Fig. 6 and Table 1).
DEAE modiand 200 mmThe Qmax aof Langmucentration of the Kd v200
mmol atrolled usin microscopy images. (a) Cellulose acetate nanobre
mat before anytylation. (c)(i) Diethylaminoethyl (DEAE) cellulose
adsorbent. (c)(ii)
cations were varied with DAECH concentrations of 50ol/g
cellulose and a repeated treatment (2 200 mmol).
nd Kd values were evaluated using the linearised formir
isotherm. However, lacking data in the low con-region of the
isotherm detracted from the reliabilityalues. Capacity was
increased from the 50 mmol todsorbent, showing that
functionalisation can be con-g DAECH amount. The 200 mmol
concentration Qmax of
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S.R. Dods et al. / J. Chromatogr. A 1376 (2015) 7483 79
Fig. 3. FTIR-AT s showCellulose acet t. (b)(C O) from ca
Fig. 4. TensileNo Press show
13 mg BSA/muncompresof Zhang etviously shocapacity hethat of
singlincrease in NaClO (Fig.ber of COO20 mmol Qmpreviously rresins,
whicThe swellincult to accocontribute thave been
Table 1Equilibrium bithe maximum
Sample
Reactant con
Qmax (mg/mKd (mg/mL) R spectra of diethylaminoethyl (DEAE) and
carboxylate (COO) cellulose adsorbentate (CA) starting material,
regenerated cellulose (RC) and DEAE cellulose adsorbenrboxylate
salt group (COONa) with increasing concentrations of NaClO applied.
strengths of diethylaminoethyl (DEAE) and carboxylate (COO)
cellulose adsorbents incrn was eight layers of uncompressed
regenerated cellulose. Error bars indicate SD.
L was similar to that we previously recorded for ansed nanobre
adsorbent [19] but was lower than that
al. [20]. Repeating the DAECH treatment has been pre-wn to
increase adsorbent capacity and the equilibriumre was increased to
27.4 mg BSA/mL, which was twicee treatment [25]. COO nanobre
adsorbents showed anQmax for increasing concentrations of oxidising
reagent,
6b and Table 1) and agrees with the increased num- groups
suggested in the FTIR spectra (Fig. 3b). Theax of 47.5 mg
lysozyme/mL was comparable to someeported values for commercially
available packed-bedh typically have exceptionally high surface
areas [29].g effect noted in the transbed pressure tests was
dif-unt for under equilibrium binding conditions and mayo
increasing capacity. Other COO nanobre adsorbentsstudied for
electrospun polyacrylonitrile where Chiu
et al. achieof a similatechniquesbinding capnanobre ain this
studof polymer ues suppormedium.
3.6. DBCs o
An AKTAmate the Dsame adsorshown to e
nding study of the varying chemical modications used to
fabricate diethylaminoethyl (D capacity of protein bound, Qmax, and
dissociation constant, Kd , estimated using a Langmu
DEAE cellulose
c. 50 mmol 200 mmol 2 200 mmol L) 7.5 13.0 27.4
0.077 0.044 0.11 ing the change in chemical groups during
adsorbent fabrication. (a) COO modication shows an increasing
height of the carbonyl peakeased with increasing compressions
applied during fabrication. The
ved an equilibrium binding capacity using lysozymerly high value
as in this study [30]. Polymer grafting
have shown advantages in vastly improving proteinacities [31].
An equilibrium capacity range using a
dsorbent was shown to be 425 times higher than thaty, also using
lysozyme and dependent on the amountgrafted [16]. These studies
reporting high capacity val-t using the high surface of nanobres as
an adsorbent
f varying chemical modications
system and custom lter holder were used to esti-BCs at 10%
breakthrough at varying owrates of thebents studied above (Fig. 7).
The residence times arexemplify how little time is required for
convective
EAE) and carboxylate (COO) cellulose nanobre adsorbents,
detailingir linear regression t.
COO cellulose
5 mmol 10 mmol 20 mmol
11.8 18.9 47.50.065 0.034 0.049
-
80 S.R. Dods et al. / J. Chromatogr. A 1376 (2015) 7483
Fig. 5. Transb lose n(b) Eight-layer orben
mass transand rangedfor the highing study a21 mg lysozadsorbents
typically infor COO andsurface area large capplateaued fbed
chroma
Fig. 6. Equilib5 MPa and mo5.3 was used fed pressures of
electrospun diethylaminoethyl (DEAE) and carboxylate (COO) cellu
DEAE and COO adsorbents at varying compressions. (c) and (d) DEAE
and COO adsfer of the target protein with a nanobre adsorbent from
4 s to 0.3 s. The highest DBCs were recordedest functionalisations
found in the equilibrium bind-nd were 12 mg BSA/mL for 2 200 mmol
DEAE andyme/mL for 20 mmol COO. At these DBCs, nanobrecompare
poorly against packed-bed media, which are
the 3085 mg/mL range for DEAE and 40100 mg/mL carboxymethyl
resins, despite suggesting reasonable
a for binding in the equilibrium study [32]. There wasacity drop
between the Qmax values and DBCs. DBCsor increasing owrates, where
in traditional packed-tography we would expect continual loss
through ow
distributionhas been padsorbent tproperties explanationlter
holdedistributiondyes. Such tstructure ofor bindingtions. Anotof
the ion-
rium binding adsorption isotherms of 8-layer electrospun
diethylaminoethyl (DEAE) andied under different reactant
concentrations. (a) BSA in 10 mM Tris buffer at pH 8 was uor COO.
Error bars indicate SD of the average Q and C values taken from
three replicatesanobre adsorbents of varying bed layers and
compressions. (a) andts of varying bed layers compressed at 5 MPa.
effects on the diffusion mass transfer. This differencereviously
reported for a non-pressed DEAE nanobreested under similar
conditions [19]. Flow distributionof the custom lter holder used
were considered as an
for the difference between Qmax values and DBCs. Ther was
developed using spacer frits for complete ow
across an adsorbent and tested visibly using colouredests would
not be able to reveal the internal microscalef nanobre matrix and
some areas may be unreachable
under ow conditions but available under static condi-her
contributing factor would be the chemical natureexchange groups.
The hydrophilic nature of the COO
d carboxylate (COO) cellulose nanobre adsorbents compressed
atsed for DEAE testing and (b) lysozyme in 20 mM acetate buffer at
pH.
-
S.R. Dods et al. / J. Chromatogr. A 1376 (2015) 7483 81
Fig. 7. Dynamic binding capacities at 10% breakthrough of
8-layer electrospun (a) diethylaminoethyl (DEAE) and (b)
carboxylate (COO) cellulose nanobre adsorbentscompressed at 5 MPa
and modied under different reactant concentrations using identical
binding conditions as before with NaCl elution. Error bars indicate
SD.
group and the swelling effect attributed to causing higher
transbedpressure may allow for greater capacity under static
conditions ifa gel-like layer was formed around nanobre strands.
However,this reasoning can only be applied to the COO adsorbents
andnot DEAE. Further investigation into the structure of
ion-exchangenanobre adsorbents is required. Convective mass
transfer mediaand particularly the non-dead-end structure of
nanobre adsor-bents, have the ability to operate at considerably
higher owrateswhich benets the overall productivity to separate
proteins [19,33].Therefore a high dynamic capacity was less
important than
attaining a repeatable capacity at higher owrates where in
largescale devices, capacity can be circumnavigated with higher
volumesof adsorbent.
3.7. DBCs of varying bed layers and compressions
DBCs at 10% breakthrough for 8-layer adsorbents compressedat
loads of 1, 5 and 10 MPa applied after electrospinning and
adsor-bents of varying bed layers (4, 8 and 12) compressed at 5
MPawere investigated for functionalisations 2 200 mmol DEAE and
Fig. 8. Dynamlayers and com(d) DEAE and Cic binding capacities
at 10% breakthrough of electrospun diethylaminoethyl (DEAE)
anpressions using identical binding conditions as before with NaCl
elution. (a) and (b) EigOO adsorbents of varying bed layers
compressed at 5 MPa. Error bars indicate SD.d carboxylate (COO)
cellulose nanobre adsorbents for varying bedht-layer DEAE and COO
adsorbents at varying compressions. (c) and
-
82 S.R. Dods et al. / J. Chromatogr. A 1376 (2015) 7483
20 mmol COO (Fig. 8). The DEAE and COO chemistry protocolswere
different in capacities with adsorbents compressed at 1
MParecording the highest DBCs at the lowest owrate tested of 900
CV/hrecording 20 mg BSA/mL and 27 mg lysozyme/mL, respectively.
Adecreasing present andThe ow disfor this initgenerally colow
transbecreate littleas a cause oDBC was fodecreasing for DEAE anthe
adsorbetein bindingall owratestrength pradequate tr
DEAE an5 MPa showbents wherrespectivelybents showsimilar
DBCdecrease andue to the ha second cocompressiothe bed vol
4. Conclus
Compresnanobre atowards theical properscale devicehence
batchthese bioseand COO mphologies, strengths wincrease wiwith no
sigbed pressuloads of DEto the hydrthe highestas a repeateand 20
mmthe lowest lthe lowest binding. At increasing However, Dat 5 and
102000 CV/h.
This studfunctionalisical to the rand operatibre
materiadevelopme
balance of capacity and material strength and tailor the
material tothe application.
Acknowledgements
thanarlysistaical S
nces
trickla. Aze
reconds BiBalasutegie485eng, Es to p. Etzejugate. Liu, Jnt for
uang
ions iniu, X.ue enhang, ctrosp480onwasent s13) 42.
Tijintillatio462ucche
enhahnol. Frey, 08) 37ardic
hum. 46 (2. Herm, 2013
Menk, et aimensh cap037a, M
U) be (200hang
of elmbranHardih pro911hang,
memmbr. SHiroteneraluloseaito, Ase ox190aito, Mizationng
TEM219a, C.
le mat Chen,luloseaito, Ars by
(200capacity from 900 CV/h to around 2000 CV/h was then DBC
appeared to stabilise for up to 12,000 CV/h.tribution through the
lter holder could be responsibleial capacity drop for increasing
owrate but DBCs weremparable for increasing owrates over 2000 CV/h.
Thed pressures of 1 MPa DEAE and COO suggested they
resistance to ow and discourage channelling effectsf the DBC
drop. A detrimental effect of compression onund for increasing
loads 5 MPa and 10 MPa with DBCsfrom 12 to 9 mg BSA/mL and 20 to 17
mg lysozyme/mLd COO, respectively. The increasing loads would
packnts more and this reduced available surface area for pro-
would lead to lower DBCs. The DBCs were stable acrosss tested
and combined with the improved mechanicaloperties, and then a loss
in DBC could be considered anade-off for large scale application.d
COO adsorbents of varying bed layers compression ated high DBCs at
10% breakthrough for the 4-layer adsor-e the bed volume was very
low at 0.07 mL and 0.09 mL,. Increasing the number of bed layers
for DEAE adsor-
ed reduced DBCs. For COO 12-layer adsorbents recordeds to
8-layer up to 3000 CV/h but then DBCs began tod may suggest that
channelling effects were presentigh transbed pressures. The bed
layer results indicatednsideration in fabricating nanobre
adsorbents wheren at 5 MPa can create a robust material but
increasingume with layers further reduced DBCs.
ions
sion and heat treatment steps during the fabrication ofdsorbents
allow their physical properties to be tunedir application as a
chromatography medium. Mechan-ties are critical for handling and
packing into larges and impact operable owrates, column capacity
and
operation time. Functionalisations also directly affectparation
properties. The differences between DEAEodications are clear as
shown by changes in mor-
transbed pressures and capacities. Absorbent tensileere similar
for DEAE and COO and were found to
th greater levels of compression after electrospinningnicant
difference between functionalisations. Trans-res show seemingly
little effect between compressingAE and yet large changes for COO,
which is attributedophilic COO groups. When studying protein
separation
attainable capacities by functionalisation were foundd treatment
of 200 mmol/g adsorbent DAECH for DEAEol/g NaClO for COO
adsorbents. Nanobres prepared atevel of compression (1 MPa) yielded
the highest DBCs atowrate, which indicates the available surface
area for5 and 10 MPa compressions capacity was decreased andbed
layers compressed at 5 MPa also decreased DBCs.BCs recorded
remained stable for increasing owrate
MPa compressions while 1 MPa was only stable above
y shows that the interactions between fabrication andation in
the synthesis of nanobre adsorbents are crit-equired physical
properties of the material for packingng a bioseparation medium.
This requires that nano-ls properties are measured and understood
alongsidents in surface chemistry, in order to strike the
correct
Wein the eand asBiolog
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Fabricating electrospun cellulose nanofibre adsorbents for
ion-exchange chromatography1 Introduction2 Materials and methods2.1
Fabricating cellulose nanofibre adsorbents2.2 DEAE modification2.3
TEMPO-mediated oxidation2.4 Morphological, chemical and tensile
strength analyses2.5 Equilibrium adsorption capacities2.6 DBCs and
transbed pressures
3 Results and discussion3.1 Surface morphology3.2 FTIR-ATR3.3
Tensile strength3.4 Transbed pressures of varying bed layers and
compressions3.5 Equilibrium absorption capacities of varying
chemical modifications3.6 DBCs of varying chemical modifications3.7
DBCs of varying bed layers and compressions
4 ConclusionsAcknowledgementsReferences