Journal of Membrane Science 318 (2008) 311316

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Journal of Membrane Science
j our nal homepage: www. el sevi er . com/ l ocat e/ memsci
Fractionation of monoclonal antibody aggregates using
membrane chromatography
Lu Wang, Raja Ghosh

Department of Chemical Engineering, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4L7, Canada
a r t i c l e i n f o
Article history:
Received 24 January 2008
Received in revised form 20 February 2008
Accepted 22 February 2008
Available online 2 March 2008
Keywords:
Monoclonal antibody
Aggregates
Campath-1H
Membrane chromatography
Hydrophobic interaction
a b s t r a c t
Monoclonal antibodies (mAbs) comprise an important class of biopharmaceuticals. Aggregation of mAbs
is a common though undesirable occurrence. Size exclusion chromatography (SEC) which is used for both
analysis and preparative separation of mAb aggregates is slow and results in poorly resolved peaks, par-
ticularly for higher order aggregates. We describe a hydrophobic interaction membrane chromatography
(HIMC) based method for rapid and efcient separation and analysis of mAb aggregates. Our method
was able to resolve Campath-1H monomer, dimer, trimer, tetramer and pentamer. The results obtained
also strongly supported our hypothesis that the hydrophobicity of mAb increases with the degree of
aggregation, i.e. a dimer is more hydrophobic than a monomer, a trimer more than a dimer, and so on.
2008 Elsevier B.V. All rights reserved.
1. Introduction
Monoclonal antibodies (mAbs) comprise an important class of
biopharmaceuticals [1] and are widely used as diagnostic agents
[2]. MAbs, being recombinant molecules which do not occur nat-
urally are less stable compared to serum antibodies. Consequently
they showsignicant tendency to self-associate into aggregates [3].
Several factors such as high concentration [4], extremes of pH [5],
high shear rates [6] and thermal stress [7] are known to encour-
age aggregate formation. In a biopharmaceutical manufacturing
process mAb aggregates can be formed during fermentation [8],
purication [5] and storage [8]. Clinically used mAbs should be
aggregate free since aggregates are immunogenic and are known
to cause adverse reactions in patients [9].
Protein-A and protein-G based afnity chromatography which
are widely used for mAb purication do not distinguish between
the antibody monomer and its aggregates [8,10] since these lig-
ands recognize and bind onto the Fc region of antibodies which
are unaffected by the aggregation process. Aggregates are therefore
separated from monomers by preparative size exclusion chro-
matography (SEC) while the aggregate content of mAb samples
is determined either by analytical SEC or by native polyacry-
lamide gel electrophoresis (PAGE) [8]. SEC is time-consuming
and cannot resolve higher order aggregates. Native PAGE is able

Corresponding author. Tel.: +1 905 525 9140x27415; fax: +1 905 521 1350.
E-mail address: rghosh@mcmaster.ca (R. Ghosh).
to resolve higher order aggregates but is extremely slow. In a
recent paper we discussed the separation of hIgG1-CD4 monomer
from its dimer using a hydrophobic interaction membrane chro-
matographic (HIMC) technique [11]. This technique which utilized
the difference in hydrophobicity between the mAb monomer
and its dimer was fast and gave signicantly better resolution
of peaks than SEC. In a more recent paper the use of column
based hydrophobic interaction chromatography for monoclonal
antibody aggregate removal has been discussed [12]. Our current
work attempts the separation of higher order mAb aggregates
such as trimer, tetramer and pentamer using HIMC. This is
based on our hypothesis that the hydrophobicity of mAb would
increase with degree of aggregation since aggregate formation
is known to take place through interactions between the rel-
atively hydrophilic Fab regions of the IgG1 type mAbs [10].
Researchers have shown that the Fc region is more hydrophobic
than the Fab regions [13]. The Fc region of the mAb is neither
involved in aggregate formation nor is affected by the process
[10].
The model mAb used in our current study is Campath-1H (also
known as Alemtuzumab). This is a humanized IgG1 type mAb
against human leukocyte antigen CD52 [14].Campath-1H is syn-
thesized by grafting hypervariable loops specic for CD52 onto a
humanantibody framework. Campath-1His usedfor the treatment
of multiple sclerosis, organ transplant rejection [8] and several
types of leukemia [15]. We examined four types of polyvinylidine
uoride (PVDF) membranes for their mAb binding properties and
selected the most appropriate among these for the HIMC experi-
0376-7388/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.memsci.2008.02.056
312 L. Wang, R. Ghosh / Journal of Membrane Science 318 (2008) 311316
ments. The working principle was that the mAb and its aggregated
forms would bind reversibly on the selected membrane in the pres-
ence of high anti-chaotropic salt concentration and these would be
eluted out in order of increasing hydrophobicity on application of a
negative salt concentrationgradient. The fractionatedsamples thus
obtained were analyzed by SEC and native PAGE to determine their
identity. The separation process was optimized for high resolution.
2. Materials and methods
2.1. Materials
Humanized monoclonal antibody Campath-1H monomer
(batch no. 44) and Campath-1H dimers (batch no. 23) (note:
the term dimers denotes a mixture of mAb dimer and
higher order aggregates; this term is commonly used in the
industry) were kindly donated by the Therapeutic Antibody Cen-
tre, University of Oxford, UK. Polyvinylidene uoride (PVDF)
microltration membranes (hydrophilic 0.1m pore size, cata-
logue # VVLP09050; hydrophilic 0.22m pore size, catalogue
# GVWP14250, hydrophilic 0.65m pore size, catalogue #
DVPP04700; and hydrophobic 0.1m pore size, catalogue #
VVHP04700) used as chromatographic media were purchased from
Millipore, USA. All samples and mobile phases used in the chro-
matographic experiments were prepared using 20mM sodium
phosphate buffer (pH 7.0) which in turn was prepared using ultra-
pure water (18.2Mcm) obtained from a Diamond Nanopure
water purication unit (Barnstead, USA). All chemicals used in the
experiments, e.g. sodium phosphate (mono- and di-basic), ammo-
nium sulfate and sodium chloride were purchased from Sigma
Aldrich, Canada.
2.2. Contact angle measurement
The suitability of the different PVDF membranes for carrying
out HIMC was assessed by contact angle measurement. The contact
angles obtained with water and 1.5M ammonium sulfate solution
in water were determined by using a DSA1 Drop Shape Analysis
system (Kruss, USA) under sessile drop measurement mode, the
drop volume being 0.02mL. The data obtained was recorded by
DSA1 Drop Shape Analysis software.
2.3. mAb Binding and elution experiments
The suitability of the PVDF membranes for HIMC was further
assessed using mAb binding and elution experiments carried out
using anAKTAPrime liquidchromatography system(GEHealthcare
Life Sciences, Canada). Two membrane discs (effective diame-
ter =18mm) were housed in a customdesigned membrane module
[16] which were then integrated with the AKTA Prime system. The
absorbance at 280nm wavelength and conductivity of the efu-
ent from the module were continuously measured and the data
was logged into a computer using Prime View (GE Healthcare Life
Sciences, Canada). The mAb was bound to the membrane from a
pulse of injectedsample inthe presence of 1.5Mammoniumsulfate
containing buffer and eluted using ammonium sulfate free buffer.
These experiments were carried out at 3mL/min ow rate. The
amounts of protein bound on the membrane and that subsequently
recovered were determined fromthe area under the curve data for
the unbound and eluted peaks, based on appropriate calibration.
2.4. Non-reducing native PAGE
Samples were tested for presence of mAb aggregates by acidic
non-reducing native polyacrylamide gel electrophoresis [8] (PAGE)
(10% gel) using a Hoefer MiniVE vertical electrophoresis unit (GE
Healthcare Life Sciences, Canada). The acidic pH ensured that the
mAb and its aggregates were all positively charged and the gels
were run with reversed polarity at 140V (40mA current). The
gels were stained with Coomassie blue dye to visualize the protein
bands.
2.5. Reducing SDS-PAGE
Campath-1H monomer and dimers were analyzed by reducing
SDS-PAGE (12.5% gel) using a Hoefer MiniVE vertical electrophore-
sis unit (GE Healthcare Life Sciences, Canada) after treatment with
dithiothreitol (DTT) to break the disulde bonds (SS). These gels
were run with normal polarity at 120V (35mA current). Protein
bands were visualized by staining with Coomassie blue dye.
2.6. Protein-A chromatographic analysis
Campath-1H dimers were analyzed by protein-A afnity chro-
matography using a HiTrapTM rProtein-A FF afnity column (GE
Life Sciences, Canada). The binding buffer used in this experiment
was 20mMsodiumphosphate (pH7.0) and while the eluting buffer
was 100mM sodium citrate (pH 3.0). Five hundred microliters of
dimers sample was injected and the separation was carried out at
1mL/min ow rate.
2.7. Separation of aggregates by HIMC
The analytical HIMC experiments were carried out using the
same experimental set up as used for testing mAb binding and
elution. Two membrane discs were used in these experiments.
The binding buffer contained 1.5M ammonium sulfate and the
separation was carried out at 3mL/min ow rate. Five hundred
microliters of mAb dimers (i.e. aggregate mixture) prepared in
binding buffer and having a total content of 0.12mg were injected
into the membrane module. The bound proteins were eluted out
with ammonium sulfate free buffer using a negative salt gradient,
going from 0% to 100% eluting buffer over 90mL. The preparative
HIMC experiments were carried out using a stack of 5 membrane
discs housed in the same membrane module. Five milliliters of
dimers solution prepared in the binding buffer and containing
1.05mg of total protein were injected for fractionation of different
components. The effect of gradient length on efciency of separa-
tion was examined. Samples corresponding to each of the eluted
peaks were collected and analyzed by SEC, non-reducing native
PAGE to identify the components present in these. Prior to SEC and
native PAGE, these samples were concentrated and desalted using
centrifugal ultralters (AmiconUltra30kDaMWCO, Millipore, USA,
catalogue # UFC903008).
2.8. Size exclusion chromatography
The Campath-1H dimers samples fractionated by HIMC were
analyzed by SEC with a Superdex
TM
200 gel ltration column
(10mm i.d., 300mm length, GE Life Sciences Canada, catalogue #
17-5175-01) using a HPLC system (Prepstar 218, Varian Canada).
Two hundred and fty millimolar sodium chloride solution was
used as the mobile phase at a owrate of 0.2mL/min. A100L loop
was used for sample injection. The molecular weight calibration
for SEC was performed using High Molecular Weight Gel Filtration
calibration kit (GE Life Sciences, Canada, catalogue # 28-4038-42).
The SEC data was analyzed using Varian Star 6.2 workstation soft-
ware.
L. Wang, R. Ghosh / Journal of Membrane Science 318 (2008) 311316 313
Fig. 1. Results obtained from contact angle experiments carried out with differ-
ent PVDF membranes at two solution conditions: in the absence and presence of
ammonium sulfate. The ammonium sulfate concentration used was 1.5M.
3. Results and discussion
PVDF is inherently hydrophobic but can be hydrophilized
using appropriate techniques [17]. It is common practice in the
membrane industry to refer to such hydrophilized hydrophobic
membranes as hydrophilic membranes. Hydrophilic PVDF mem-
branes have been shown to be suitable to carrying out HIMC
since they demonstrate hydrophobic properties in the presence
of high anti-chaotropic salt concentration, i.e. can bind proteins,
but are quite hydrophilic in the absence of such salts, i.e. can
release the bound proteins [18]. Fig. 1 shows the results of the
contact angle experiments carried out with the different PVDF
microltration membranes at two solution conditions, i.e. with
and without ammonium sulfate. The addition of anti-chaotropic
salts like ammoniumsulfate result in the removal of the structured
water layer adjacent to the membrane surface and accentuate their
hydrophobic properties. However, as soon as the salt is removed
the water layer recovers and the surface becomes hydrophilic. The
hydrophobic PVDF membrane with 0.1m pore size was studied
mainly for comparison. The contact angle results indicate that its
hydrophobicity did not change signicantly with salt concentra-
tion, i.e. it was hydrophobic at both solution conditions. With the
hydrophilic PVDF membranes, the contact angles increased quite
signicantlyduetothepresenceof 1.5Mammoniumsulfate, clearly
indicating that these became hydrophobic. The biggest change in
contact angle was observed with the one with 0.22m pore size.
This membrane had the smallest contact angle at the salt free con-
dition i.e. it was the most hydrophilic. However, in the presence of
salt, its contact angle was greater that the two other hydrophilic
PVDF membranes examined. These results seemed to suggest that
the membrane with 0.22mpore size was most suitable for HIMC.
Fig. 2 shows the results of the mAb binding and elution
experiments carried out with the different PVDF membranes. In
these experiments the membranes were challenged with 5mL of
0.3mg/mL Campath-1H monomer solution prepared using bind-
ing buffer i.e. having 1.5M ammonium sulfate concentration. The
hydrophobic PVDF membrane bound all the mAb fromthe injected
sample but the binding was irreversible in nature as evident from
the lack of an elution peak. Among the hydrophilic PVDF mem-
branes, the one with 0.22m pore size adsorbed the maximum
amount of mAb (based on the area of the unbound peak). Based on
the amount of Campath-1H eluted, the reversible binding capac-
ity of this membrane was estimated to be around 20mg/mL of
membrane bed volume. The monoclonal antibody recovery from
the membrane was approximately 97%, this data being obtained
by material balance. The results obtained in the mAb binding and
elution experiments were consistent with the expectations based
Fig. 2. Binding and elution of Campath-1H on different PVDF membranes (elut-
ing buffer: 20mM sodium phosphate buffer, pH 7.0; binding buffer: eluting
buffer +1.5M ammonium sulfate).
on the contact angle measurements. The hydrophilic PVDF mem-
brane with 0.22m pore size was used in all subsequent HIMC
experiments.
The Campath-1H dimers sample was analyzed by polyacry-
lamide gel electrophoresis. Fig. 3 shows the acidic native PAGE (A)
run with reversed polarity and the reducing SDS-PAGE (B) obtained
withthe mAb dimers. Five distinct bands canbe seenonthe stained
native gel, the lowest corresponding to the Campath-1Hmonomer,
and the degree of aggregation increasing toward the top of the gel.
The SDS-PAGE was run after treating the sample with dithiothre-
itol (DTT) to break the disulde bonds (SS) between the mAb
light chains andheavy chains. The stainedgel shows twobands, one
corresponding to a molecular weight of 55kDa (heavy chain) and
the other 25kDa (light chain). The SDS-PAGE results prove that the
Fig. 3. Polyacrylamide gel electrophoresis of Campath-1H dimers (A) acidic native
PAGE run with reversed polarity (B) reducing SDS-PAGE.
314 L. Wang, R. Ghosh / Journal of Membrane Science 318 (2008) 311316
Fig. 4. Protein-A afnity chromatogram obtained with Campath-1H dimers (elut-
ing buffer: 20mM sodium phosphate buffer, pH 7.0; binding buffer: eluting
buffer +1.5M ammonium sulfate).
bands observedonthe native gel were due tothe mAbandits aggre-
gates alone and no other proteins were present in the Campath-1H
dimers sample at detectable concentration.
Fig. 4 shows the protein-Aafnity chromatogramobtained with
Campath-1H dimers. All components present in the dimers, i.e.
the mAb and its aggregates bound to the afnity column at condi-
tions favorable for binding (pH 7.0) and were eluted out as a single
peak at acidic pH conditions. Protein-A binds antibodies through
their Fc region. Therefore the Fc region of the mAb aggregates were
unaffected by the aggregation process. These results are consis-
tent with reports by earlier workers [8,10] that protein-A afnity
chromatography could not be used to separate mAb from their
aggregates. Protein aggregation has been shown to be initiated by
Fig. 5. Analytical HIMC chromatogramobtained with Campath-1Hdimers (number
of membrane discs: 2; sample volume: 500L; amount injected: 0.12mg; owrate:
3mL/min; gradient length: 90mL; eluting buffer: 20mM sodium phosphate buffer,
pH 7.0; binding buffer: eluting buffer +1.5M ammonium sulfate).
Fig. 6. Preparative HIMC separation of Campath-1H dimers (number of membrane
discs: 5, sample volume: 5mL, amount injected: 1.05mg, ow rate: 3mL/min, gra-
dient length: 90mL, 100mL and 130mL).
specic interactions between folding and unfolding intermediates
[19]. Earlier workers have reported that Campath-1Hdimer forma-
tion takes place due to FabFab interaction [10]. The Fab regions
of the mAb have lower microstability [20] compared to that of
the Fc region, making them more prone to unfolding and refold-
ing and hence more likely responsible for aggregate formation. The
Fab regions of the mAb molecules involved in aggregate formation
would therefore be present towards the inside of these complexes.
Consequently the Fc regions of these molecules are more likely to
be more exposed to the outside. In a recent study it was shown that
Fig. 7. SEC chromatograms obtained with the dimers (feed) and fractionated sam-
ples obtained by preparative HIMC using 90mL negative salt gradient.
L. Wang, R. Ghosh / Journal of Membrane Science 318 (2008) 311316 315
when an IgG molecule bound to a PVDF membrane via hydropho-
bic interaction, it still retained the ability to bind its antigen [21].
This clearly showed that the antigen binding Fab regions were not
involved in the membrane binding process which presumably took
place through the more hydrophobic Fc region. Nagaoka et al. [13]
have also shown that IgG binding onto hydrophobic polymer sur-
faces took place through the Fc region [13] and concluded that this
region was more hydrophobic than the Fab region. Based on these
observations we hypothesized that as the degree of mAb aggrega-
tion increased, a greater proportion of the external surface area of
these complexes would be occupied by the Fc region. As a result
the hydrophobicity would increase with increase in degree of mAb
aggregation. To test this hypothesis, we carried out HIMC experi-
ments with the dimers.
Fig. 5 shows the analytical HIMC chromatogram obtained with
a stack of two hydrophilic PVDF membrane discs (having 0.22m
pore size) by injecting 500L of Campath-1H dimers (having a
total protein content of 0.12mg). The purpose of this experiment
was to test the feasibility of resolving the different mAb aggregate
basedonhydrophobicitydifference: themorehydrophobic thepro-
tein, the later it would appear on the negative salt gradient. The
chromatogramshows 5 distinct peaks and a shoulder, possibly cor-
responding to the monomer, dimer, trimer, tetramer, pentamer and
hexamer respectively. The amount of protein injected in the ana-
lytical HIMC experiment was quite low and this did not allow for
sufcient protein samples to be collected from each of the peaks
for identity determination. In order to do this, preparative HIMC
experiments were carried out using the same membrane module
with more membrane discs.
Fig. 6 shows the preparative HIMC chromatograms obtained
with three different negative salt gradients. In each of these exper-
iments 5mL of mAb dimers (containing 1.05mg of total protein)
prepared in binding buffer was injected into the membrane mod-
ule which contained ve membrane discs. This was followed by
the application of the appropriate negative salt gradient to elute
the mAb and its aggregates in order of increasing hydrophobicity.
In each of the chromatograms, ve distinct peaks presumable due
to monomer, dimer, trimer, tetramer and pentamer respectively
can be observed. The retention times of the peaks increased but
the peak resolution did not get signicantly better with increase in
gradient length. In fact the rst peak became quite broad with the
130mL gradient, this being a shallowgradient. The peaks obtained
with the 90mL gradient were quite well resolved and therefore
collected for identity determination using SEC and native PAGE.
Fig. 7 shows the SEC chromatograms obtained with the feed
(dimers) and samples from each of the resolved peaks obtained
Fig. 8. Acidic, non-reducing native PAGE results obtained with dimers and HIMC resolved peaks. (Dimers: lanes B, C and F, peak 1: lane A, peak 2: lane D, peak 3: lane E, peak
4: lane H and peak 5: lane G).
316 L. Wang, R. Ghosh / Journal of Membrane Science 318 (2008) 311316
Table 1
Estimation of molecular weight of the HIMC peak components
HIMC peak # SEC Retention time (min) Predicted molecular weight
1 57.1 208kDa
2 47.5 489kDa
3 43.5 644kDa
4 39.3 Out of calibration range
5 38.7 Out of calibration range
Calibration molecular weight range 10669kDa. Calibration equation:
Mw=0.7611t
2
108.98t +3947.2, where t is retention time (min) and Mw is
molecular weight (kDa), R
2
=0.985.
from the preparative HIMC experiment with 90mL negative salt
gradient. Quite clearly SEC was able to separate the mAb monomer
from its aggregates (see the feed chromatogram). However, each
aggregate type could not be resolved by SEC. In fact, the dimers
chromatogram shows only three overlapping aggregate peaks and
not four as could be expected based on the preparative HIMC
results. The SEC retention times obtained with the HIMC fraction-
ated samples showed an inverse relationship with the HIMC peak
retention times, i.e. the molecular weight of the HIMC peak com-
ponents increased with increase in retention time. This provided
preliminary evidence supporting our hypothesis, i.e. hydrophobic-
ity increased with degree of aggregation. In order to verify that
the fractionated samples indeed contained the monomer, dimer,
trimer, tetramer and pentamer respectively, the SEC peak reten-
tion times were compared with molecular weight calibration data
(see Table 1). Based on the calibration equation shown in the table
the molecular weights of the proteins present in the rst, sec-
ond and third HIMC peaks were estimated to be 208kDa, 489kDa
and 684kDa respectively. The molecular weight of IgG1 type mAb
monomer is in the 150160kDa range and therefore the dimer
and trimer should have molecular weights in the 300320kDa and
450480kDa ranges respectively. The main reason for this discrep-
ancy is that the calibration proteins were globular while mAb and
its aggregates hadirregular shapes whichmadeit harder for themto
penetrate the pores of the SEC medium[3]. Consequently they had
lower retention times than globular proteins having similar molec-
ular weights. The molecular weights of the mAb and its aggregates
were therefore overestimated by SEC. It is therefore safe to say that
the rst three HIMC peaks were due to mAb monomer, dimer and
trimer respectively. The fourth and fth HIMC peaks were presum-
ably due to tetramer and pentamer but this could not be veried
by SEC on account of their retention times being out of range.
Fig. 8 shows the acidic non-reducing native-PAGE results
obtained with the dimers and samples from the fractionate peaks
obtained by preparative HIMC. The dimers sample was loaded
on lanes B, C and F. The samples from HIMC peaks 15 were
loaded in lanes A, D, E, H and G respectively. The dimers sam-
ple showed ve distinct peaks corresponding to the monomer,
dimer, trimer, tetramer andpentamer respectively. Quiteclearlythe
major components present in lanes A, D, E, H and G were the mAb
monomer, dimer, trimer, tetramer and pentamer respectively. The
HIMC technique was therefore able to resolve Campath-1H from
its aggregates. Each individual aggregate type from dimer to pen-
tamer was resolved. These results provide further evidence that the
hydrophobicity of mAb increased with degree of aggregation.
4. Conclusion
The HIMC technique discussed in this paper was more efcient
than SEC both in terms of speed and resolution. It was able to
resolve Campath-1H monomer, dimer, trimer, tetramer and pen-
tamer as separate peaks. This study also clearly demonstrated that
hydrophobicity of mAb increased with degree of aggregation, i.e. a
trimer was more hydrophobic than a dimer and so on. Amethod for
selecting appropriate membrane for HIMC based on contact angle
measurement was described. The conclusions drawn based on con-
tact angle measurements were in excellent agreement with the
results obtained from the mAb binding and elution experiments.
Acknowledgements
We thankDrs. Geoff Hale, PruBird, MarkFrewinandother mem-
bers of the Therapeutic Antibody Centre, Oxford University, UK
for donating the Camapth-1H monomer and dimers samples. Wei
Chen of Chemical Engineering Department, McMaster University
is acknowledged for helping with the contact angle measurement
experiments. We thank Natural Science and Engineering Research
Council of Canada (NSERC) for funding this study. LW thanks Shell
Canada and China National Scholarship Council for personal schol-
arships. RG holds the Canada Research Chair in Bioseparations
Engineering.
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