-
Effect of PEGylation on the Solution Conformationof Antibody
Fragments
YANLING LU,1 STEPHEN E. HARDING,1 ALISON TURNER,2 BRYAN SMITH,2
DILJEET S. ATHWAL,2
J. GÜNTER GROSSMANN,3 KENNETH G. DAVIS,1 ARTHUR J. ROWE1
1National Centre for Macromolecular Hydrodynamics, University of
Nottingham, Sutton Bonington LE12 5RD, England, UK
2UCB-Celltech, 216 Bath Road, Slough, Berkshire SL1 1NH, UK
3CCLRC Daresbury Laboratory, Synchrotron Radiation Department,
Warrington, Cheshire WA4 4AD, England, UK
Received 16 March 2007; revised 18 May 2007; accepted 13 June
2007
Published online in Wiley InterScience
(www.interscience.wiley.com). DOI 10.1002/jps.21170
Corresponde115-951-6148; FE-mail: steve.ha
Journal of Pharm
� 2007 Wiley-Liss
2062 JOURN
ABSTRACT: Covalent attachment of poly(ethylene glycol) (PEG) to
therapeutic anti-body fragments has been found effective in
prolonging the half-life of the proteinmolecule in vivo. In this
study analytical ultracentrifugation (AUC) in combinationwith small
angle X-ray scattering (SAXS) has been applied to a number of
antibodyfragments and to their respective PEGylated conjugates.
Despite the large increase inmolecular weight due to the attachment
of a 20–40 kDa PEG moiety, the PEGylatedconjugates have smaller
sedimentation coefficients, s, than their parent antibodyfragments,
due to a significant increase in frictional ratio f/fo (from �1.3
to 2.3–2.8):the solution hydrodynamic properties of the conjugates
are clearly dominated by thePEG moiety ( f/fo �3.0). This
observation is reinforced by SAXS data at high values of
r(separation of scattering centres within a particle) that appear
dominated by the PEGpart of the complex. By contrast, SAXS data at
low values of r suggest that there are nosignificant conformational
changes of the protein moiety itself after PEGylationThe location
of the PEGylation site within the conjugate was identified, and
found tobe consistent with expectation from the conjugation
chemistry. � 2007 Wiley-Liss, Inc. andthe American Pharmacists
Association J Pharm Sci 97:2062–2079, 2008
Keywords: PEGylation; immunoglobul
in; antibody fragment; conformation
INTRODUCTION
Biomedical exploitation of the discriminationand affinity of
antibody binding properties hasbeen advanced by biotechnological
developments.There are now 18 commercially available mono-clonal
antibody products and more than 100 inclinical development. In the
near future, engineer-ed antibodies are predicted to account for
>30%
nce to: Stephen E. Harding (Telephone: þ44-0-ax:
þ44-0-115-951-6142;[email protected])
aceutical Sciences, Vol. 97, 2062–2079 (2008)
, Inc. and the American Pharmacists Association
AL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 6, JUN
of all revenues in the biotechnology market.1
High-level expression systems have been develop-ed for mammalian
cell expression of antibodies;however, the large quantities needed
in thera-peutic doses make them prohibitively costly.Antibody
fragments (Fab’, Fv and scFv)2–4 andengineered variants (diabodies,
triabodies, mini-bodies and single-domain antibodies)1,5–7 that
canbe readily produced in large-scale in microbialsystems are now
emerging as credible alternativesfor in vitro immunoassays and in
vivo tumour-targeting therapy.8
The improved pharmacokinetics associated withantibody fragments
in tumour penetration areoften modulated by the tendency of such
fragments
E 2008
-
SOLUTION CONFORMATION OF ANTIBODY FRAGMENTS 2063
to have very short circulation times in vivo.6
However, enhancement of the pharmaceuticalproperties of antibody
fragments by ‘‘PEGylation’’,or attachment of poly(ethylene glycol)
(PEG) to thefragment, appears to confer a prolonged
circulatinghalf-life on the antibody fragments.9–13
ProteinPEGylation modulates many properties of bio-medical
significance, including reduced toxicity,reduced immunogenicity and
antigenicity; slowingrates of clearance and proteolysis, and
enhancingsolubility and stability (for reviews, see Refs.
14,15).
The molecular basis of the beneficial effects ofPEGylation of
therapeutic proteins can be attri-buted to the unique
physicochemical properties ofPEG itself (for reviews, see Refs.
16,17). One of themost distinct features of PEG is its propensity
tooccupy a large volume in an aqueous environmentthrough
time-averaged water association and pre-sents a volume 5–10 times
larger than a solubleprotein of comparable molecular
weight,11,14,16,18,19–21
and this can confer on a protein conjugated to itadvantageous
features.
Numerous functionalised PEG molecules arenow available
commercially.14,18,22 An appro-priate choice of the size and
structure of thePEG moieties and the conjugation chemistry hasbeen
found crucial to balance the desired pro-longed half-life in vivo,
while maintaining anacceptable level of relevant biological
activity oractivities (including not only antigen binding
andantibody effector function, but also the ability tolocalise to
certain tissues such as tumours).10
Hence, the molecular weight of the PEG moiety,its structure, the
number and location of the
Figure 1. Schematic representation of an infragments. (a) IgG;
(b) (Fab’)2; (c) Fab: no hingare linked through one disulfide bond;
(d) Facysteine.
DOI 10.1002/jps J
PEG moieties attached to the protein, as wellas the chemical
method of attachment, alldetermine the physicochemical and
pharma-cological properties of the resulting
proteinconjugate.10,23
Currently there are two different kinds ofPEG structure—linear
or branched—which canbe attached to proteins. A branched structure
witha single attachment site could be more favourablethan the
linear structure since a high molecularweight PEG moiety can
apparently be obtainedwithout increasing the number of
attachmentsites.24,25 Moreover, branched chain PEGylatedproteins
have been found to be more stable againstenzyme proteolysis than
linear moieties.23,25
Mono-site attachment of a limited number oflonger chain PEG
molecules rather than multi-site attachment of a greater number of
smallerPEG molecules has been found most beneficial forretaining a
higher level of biological activity.9,10
Generation of PEGylated protein requires site-specific
conjugation strategies to minimise theamounts of unmated isomers
produced.19,23,26 Inthe case of Fab’ (Fig. 1) it is possible to
engineer acysteine residue in the ‘residual’ hinge region ofthe
molecule (or more than one if required): tofacilitate specific
PEGylation through reactionbetween the thiol group of the cysteine
and themaleimide group present in a PEG-maleimideconstruct to form
a stable thioether linkage.9,10,24
This reaction can generate site-specific PEGyla-tion of the Fab’
(Fig. 2) resulting in yielding ahomogeneous PEGylated product
preserving thefull binding activities of the antibody
fragment.9
tact antibody molecule and some antibodye region, the heavy
chain and light chainb’: has a short hinge region with a free
OURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 6, JUNE 2008
-
Figure 2. Schematic illustration of site-specific Fab’
PEGylation. PEG-maleimidereacts with the thiol group in the hinge
region of a Fab’ fragment forming a stablethioether linkage.
2064 LU ET AL.
Whilst the pharmacokinetics and pharma-codynamics of PEGylated
proteins have beenextensively studied, their solution properties
arenot as well characterised. Using thiol/maleimideconjugation
chemistry a group of PEGylatedantibody products has been generated
to allowinvestigation of their solution properties and toassess if
PEGylation has influenced proteinconformation.
The samples were characterised using thecomplementary probes of
analytical ultracentri-fugation (AUC) and small angle X-ray
scattering(SAXS). AUC can tell us about the overallhydrodynamic
properties of the complexes andwhether, from measurement of the
sedimentat-ion coefficient and the translational frictionalratio
whether it is the protein or PEG componentswhich dictate the
properties.27,28 This techniqueoffers significant advantages
compared to chro-matographic based methods for the study ofcomplex
systems like these in that it coversa very wide range of molecular
size without anyrequirement for known standards for
calibration.There are no losses arising from macromolecule/gel
matrix interactions caused by macromoleculesirreversibly binding to
chromatographic media, orfrom the failure of the conjugate
molecules to be‘‘electrophoresed’’ into polyacrylamide gels.
Another powerful tool we employ in thisstudy is small angle
SAXS, which probes thedistribution of SAXS density in the
scatteringparticle (see, e.g., 29,30), allowing deductions to
bemade about the conformation of the scatteringparticles, and in
particular if the effect of com-plexation changes the conformation
of the proteincomponent in anyway. By combining
ultracen-trifugation with SAXS, it is possible to make anassessment
of the effect of PEGylation on themolecular integrity of a
selection of antibodyfragments.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 6, JUNE
2008
MATERIALS AND METHODS
Production and Purification of Antibody Fragment
Three antibody fragments, human g1 Fab’; humang1 F(ab’)2 and
murine g1 Fab’ and associatedPEGylated products were selected for
study.
Human g1 Fab’ was derived from E. coli: cellslurry was extracted
in 0.1 M Tris/10 mM EDTA,pH 7.4 for 16 h at 308C with constant
agitation.The cells were removed by centrifugation at10,000 rpm,
for 30 min, followed by 0.22 mmfiltration and adjustment to pH 7.0
using 2MTris-HCl, pH 8.5. The Fab’ was purified byapplication of
the clarified periplasmic extractto a GammaBind G (GE-Healthcare,
Chalfont St.Giles, UK) column previously equilibrated withphosphate
buffered saline (PBS). The boundproduct was eluted with 0.1 M
glycine HCl, pH2.7, then neutralised with 2 M Tris-HCl, pH 8.5.The
Fab’ was quantified by absorbance at 280 nm,concentrated four-fold
via ultrafiltration (UF,10 kDa molecular weight cut-off membrane)
andbuffer exchanged into 20 mM Tris-HCl, pH 8.0.The diafiltered
product was applied to a pre-equilibrated (20 mM Tris-HCl, pH 8.0)
anionexchange column, Poros 50HQ (Applied Biosys-tems, Warrington,
UK). The Fab’ was collected inthe unbound fraction, concentrated
and bufferexchanged into 100 mM sodium phosphate buffer,pH 6.0
containing 2 mM EDTA, to a stock con-centration of 19.5 mg/mL.
Human g1 F(ab’)2 was obtained by pepsin digestof IgG and
purified by gel filtration (S-200HRAmersham Pharmacia Biotech,
Little Chalfont,UK). The product was concentrated and
bufferexchanged into 100 mM sodium phosphate buffer,pH 6.0
containing 2 mM EDTA, to a stockconcentration of 21.0 mg/mL.
Murine g1 Fab’ was mammalian derived using aNS0 cell line, grown
in serum-free conditions in a
DOI 10.1002/jps
-
SOLUTION CONFORMATION OF ANTIBODY FRAGMENTS 2065
100 L fermenter. The fermenter supernatantwas concentrated 10
times and then applied to apreequilibrated (20 mM sodium
phosphate/150 mM sodium chloride, pH 7.1) GammaBindPlus Sepharose
(GE-Healthcare) column. TheFab’ was eluted with 0.1 M glycine-HCl,
pH 2.8,concentrated and buffer exchanged into 100 mMsodium
phosphate buffer, pH 6.0 containing 2 mMEDTA, to a stock
concentration of 19.6 mg/mL.
The concentrations of the protein solutions weredetermined by
measuring absorbance at 280 nmwith extinction coefficients
calculated from theamino acid compositions.
PEGylation of the Fab’ fragments was achievedby site specific
attachment of PEG-maleimidederivatives purchased from Nektar
Therapeutics(Mountain View, CA) and NOF Corporation(Tokyo, Japan).
to selectively reduced thiols.The PEGylated Fab’ products used in
this studyare illustrated in Figure 3. hFab’-(L)PEG25k(Fig. 3a) was
prepared by the reduction of humang1 Fab’, in 100 mM sodium
phosphate buffer/2mM EDTA, pH 6.0, using
2-mercaptoethylamine(2-MEA) at a final concentration of 5 mM for30
min at 378C. The reductant was removed viadiafiltration (10 kDa
molecular weight cut-off)into 100 mM sodium phosphate buffer/2
mMEDTA, pH 6.0. The resultant thiol was titrated
Figure 3. Schematic illustration of (a) (hF20k); (c)
(mFab’-(L)PEG2� 20k); (d) (DFM-the general structure for
monomethoxy PEG
DOI 10.1002/jps J
using 4,40-dithiodipyridine (DTDP) by measure-ment of the
thiopyridine released at 324 nm; aratio of approximately one thiol
per Fab’ fragmentwas obtained. A mono-maleimide PEG derivativewith
a 25 kDa linear PEG chain was added to thereduced Fab’ using a
molar ratio of 1.0:2.4 (Fab’/PEG) and incubated for 18 h at ambient
tem-perature. The Fab’–PEG mixture was thenadjusted to pH 4.5 by
the addition of glacial aceticacid and applied to a preequilibrated
(50 mMsodium acetate, pH 4.5) cation exchange column,SP Sepharose
HP (Amersham Pharmacia Bio-tech). The product was eluted using a
lineargradient of 0–250 mM sodium chloride over 20column volumes
(CV) then concentrated andbuffer exchanged using a stirred cell
(Amicon;10 kDa molecular weight cut-off membrane) into50 mM sodium
chloride, 125 mM sodium chloride,pH 5.5 to a final concentration of
4.8 mg/mL, asmeasured by the absorbance at 280 nm. The finalproduct
was characterised by sodium dodecylsulphate–polyacrylamide
electrophoresis (SDS–PAGE) and gel filtration–high performance
liquidchromatography (GF–HPLC).
hFab’-(Br)PEG2� 20k (Fig. 3b) was preparedas described for
hFab’-(L)PEG25k, except that abranched mono-maleimide PEG
derivative (con-sisting of two linear 20 kDa chains) of overall
size
ab’-(L)PEG25k); (b) (hFab’-(Br)PEG2�(Br)PEG2� 20k).
CH3–(CH2CH2O)n– is(mPEG).
OURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 6, JUNE 2008
-
2066 LU ET AL.
40 kDa was used as the PEGylation reagent. Thefinal product was
presented in 50 mM sodiumacetate, 125 mM sodium chloride, pH 5.5 to
aconcentration of 18 mg/mL and characterised bySDS–PAGE and
GF–HPLC.
mFab’-(L)PEG2� 20k (Fig. 3c) was prepared byreduction of murine
g1 Fab’ in 100 mM sodiumphosphate buffer, pH 6.0 containing 2 mM
EDTAusing tris (hydroxypropyl)phosphine (THP,0.5 Min water, Cytec,
Bradford, UK) at a finalconcentration of 2.5 mM for 1 h at
ambienttemperature. The reductant was removed viadiafiltration (10
kDa molecular weight cut-off)into 100 mM sodium phosphate buffer,
pH 5.7containing 2 mM EDTA. Titration of the gener-ated thiols with
DTDP resulted in approximatelytwo thiols per Fab’ molecule due to
the reductionof the interchain disulfide bond between the heavyand
light chain of the Fab’. The reduced Fab’ wasincubated with a
mono-maleimide PEG derivativewith a 20 kDa linear PEG, at a molar
ratio of1.00:3.25 (Fab’/PEG) at ambient temperature for18 h. This
resulted in a product where two 20 kDaPEG chains were attached to
the Fab’ via the thiolat the C terminal end of the heavy chain
andthe light chain. Hence, there was no interchaindisulphide bridge
between the heavy and lightchains. The Fab’–PEG mixture was then
adjustedto pH 4.5 by the addition of glacial acetic acid andapplied
to a preequilibrated (50 mM sodiumacetate, pH 4.5) SP Sepharose HP
column. Theproduct was eluted using a linear salt gradient, of0–125
mM sodium chloride over 20 CV, concen-trated and buffer exchanged
into 50 mM sodiumacetate, 125 mM sodium chloride, pH 5.5 to a
finalstock concentration of 31.7 mg/mL. The finalproduct was
characterised by SDS–PAGE andGF–HPLC.
DFM-(Br)PEG2� 20k (Fig. 3d): The reductionwas performed as
described for hFab’-(L)PEG25 k.PEGylation was achieved by
incubation of abis-maleimide derivative of a branched PEG
Table 1. Molecular Weights M and PartiaAntibody Fragments
Sample Attached PEG (kDa
Murine g1 Fab’ 0Human g1 Fab’ 0Human g1 F(ab’)2 0mFab-(L)PEG2�
20k 40hFab’-(L)PEG25 k 25hFab’-(Br)PEG2� 20k 40DFM-(Br)PEG2� 20k
40
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 6, JUNE
2008
(composed of two 20 kDa linear chains) with thereduced Fab’
using a molar ratio of 2.2:1.0 (Fab’/PEG) for 18 h at ambient
temperature. ThediFab’–PEG mixture was purified as describedfor
hFab’-(L)PEG25 k, but using a linear saltgradient of 0–250 mM
sodium chloride over 30 CV.The final product was concentrated and
bufferexchanged into 50 mM sodium acetate, 125 mMsodium chloride,
pH 5.5 to a final concentration of13.5 mg/mL and was characterised
by SDS–PAGEand GF–HPLC.
Concentrations of the PEGylated protein solu-tions were measured
from measurement of ultra-violet (UV) absorbance at a wavelength of
280 nm.The extinction coefficient of the PEG is negligibleas pure
PEG revealed a minimal signal contribu-tion,11 therefore a
weight-averaged extinctioncoefficient was used for each PEGylated
moleculeby taking into account the contribution of themass of the
PEG to the conjugate. For instance, inthe case of mFab-(L)PEG2�
20k, at 280 nm theextinction coefficient of the Fab’ alone is
1614mL g�1 cm�1, and taking into account the masscontribution of
the 40 kDa PEG, the weight-averaged extinction coefficient for
mFab-(L)PEG2� 20k is 873 mL g�1 cm�1.
The commercial manufacturers claim for themolecular weight of
the branched PEG (2� 20 k)of 40 kDa was checked by both size
exclusionchromatography coupled to multi-angle laser
lightscattering (courtesy of Dr. G. Morris, NCMH)yielding a value
for the weight average molecularweight Mw of (40.5� 1.2) kDa.
Table 1 lists the PEGylated conjugates usedin this study. The
partial specific volume n ofeach antibody molecule was calculated
usingthe routine SEDNTERP31 from the amino acidcomposition.
Following Lepori and Mollica32 andNichol et al.,33 a value of 0.83
mL/g was usedas the partial specific volume for the all thePEG
molecules evaluated. The weight-averagedpartial special volume for
each of the PEGylated
l Specific Volumes n of PEGylated
) Molecular Weight M (kDa) n (mL/g)
47 0.72648 0.72696 0.72687 0.77473 0.76288 0.777
136 0.759
DOI 10.1002/jps
-
SOLUTION CONFORMATION OF ANTIBODY FRAGMENTS 2067
conjugates (see Table 1) was then calculated usingthe following
relation:34
nc ¼XNi¼1
fini ¼ fpnp þ fnpnnp (1)
where fp and fnp are the weight fractions of proteinand
nonprotein components respectively, and npand nnp their partial
specific volumes.
SDS–PAGE and HPLC Analysis of PEGylatedAntibody Fragments
The Fab’ fragments and their associated PEGy-lated products were
analysed for purity by SDS–PAGE and GF–HPLC prior to the
hydrodynamicstudies.
SDS–PAGE was performed using Novex Tris-Glycine (4–20%) gels
(Invitrogen EC60255BOX,Paisley, UK) under both nonreducing and
redu-cing conditions. For nonreducing conditions 10 mLof a 0.4
mg/mL solution of the sample was diluted1:1 with Tris-Glycine SDS
Sample buffer x2(Invitrogen LC2676) and boiled for 2 min;
forreducing conditions 2.2 mL of NuPage SampleReducing Agent� 10
(Invitrogen NP0004) wasincluded in the nonreducing mixture and
boiledfor 3 min. In both cases 10 mg was loaded onto thegel and ran
according to the manufacturer’sinstructions (a constant voltage of
125 V for 90min). The bands on the gel were visualised byCoomassie
Blue staining.
GF–HPLC was performed using an HPLCsystem (Agilent 1100,
Stockport, UK) with ZorbaxGF450 (Agilent 884973-902) and Zorbax
GF250(Agilent 884973-901) columns connected in series.The elution
buffer was 0.2M sodium phosphate,pH 7.0 containing 10% ethanol. The
sampleswere analysed at 1.0 mL min�1 isocratically with a30 min run
time, at a wavelength of 280 nm.
Sedimentation Velocity and Equilibrium in theAnalytical
Ultracentrifuge
All Fab’ fragments and their associated PEGy-lated products were
diluted to between 0.5 and1.6 mg/mL at six different concentrations
prior tosedimentation velocity experiments in an OptimaXLA (Beckman
Instrument, Palo Alto, CA), at208C using an An60Ti four-place rotor
running atvarious rotor speed (45000 rpm for velocity runsand
12000–14000 rpm for equilibrium runs). Thesedimentation process was
monitored by UV
DOI 10.1002/jps J
absorbance with a scan interval of 4 min, withthe buffer as the
reference solvent. Absorptionoptics was chosen for studying
PEGylated anti-bodies as PEG had minimal signal contribution at280
nm11: any unconjugated PEG would not bedetected.
We also investigated the sedimentation coeffi-cient behaviour of
the linear ‘‘20 kDa’’ (molecularweight estimated by the
manufacturer) PEG re-agent (see Fig. 3b) and the branched ‘‘40
kDa’’ PEGreagent (see Fig. 3c) using the interference opticalsystem
on an Optima XLI (Beckman Instrument).The 20 and 40 kDa PEG samples
(in powder form)were dissolved separately in 50 mM sodium
acetate,125 mM sodium chloride buffer (pH5.5) andconcentrations
were determined using an AtagoDD-5 differential refractometer
(Jencons Scien-tific, Leighton Buzzard, UK) (dn/dc¼ 0.134
mL/g).Samples were then diluted to between 0.3 and2.3 mg/mL prior
to the velocity run at 50000 rpm.
Sedimentation velocity data were analysed bythe so-called least
squares g�(s) or ‘‘ls-g�(s)’’procedure as implemented in
SEDFIT.35,36 g�(s)profiles confirmed the monodispersity and
theaggregate-free nature of the solutions (see Dis-cussions later),
which are critical for the sub-sequent interpretation of the SAXS
data. Theexperimental sedimentation coefficient valuess20,b (the
designation s20,b to indicate that thevalue reported applies to
conditions of 20.08C in asolvent b, which in this case is the
formulationbuffer) obtained from SEDFIT have been cor-rected to
s20,w (standard solvent conditions—thedensity and viscosity of pure
water at 20.08C)using SEDNTERP.31 The s20,w values obtained
atdifferent concentrations were extrapolated to zeroconcentration
to yield s020;w thereby eliminatingsolution nonideality
effects.37
Sedimentation equilibrium data were evaluatedby the
model-independent routine MSTARA38—no model (ideal, single solute,
etc.) has to beassumed a priori, as required by other
software.Determination of the baseline absorption byover-speeding
the centrifuge was necessary tocorrect for UV-absorbance from
nonmacromole-cular material. At each loading concentration,
theMSTARA program provides a plot of M�(r) versusj(r) for
determination of molecular weight, whereM�(r) is an operational
point average molecularweight and j(r) is the radial displacement
squaredparameter defined by:
jðrÞ ¼ ðr2 � r2aÞ
r2b � r2a(2)
OURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 6, JUNE 2008
-
2068 LU ET AL.
where ra, rb are the radial positions of the cellmeniscus and
cell base, respectively. Althoughthe M�(r) values themselves have
no physicalmeaning, the M�(r) extrapolated to the cellbase (j(r)¼
1) yields the apparent weight-averagemolecular weight over all the
macromolecularcomponents in the ultracentrifuge cell, Mw.
39 Mwat different concentrations were extrapolated tozero
concentration to eliminate any contributionfrom thermodynamic
nonideality.37
Small Angle X-Ray Scattering (SAXS)
The SAXS data were obtained at Station 2.1 at theSynchrotron
Radiation Source (SRS, Daresbury,UK), employing sample-to-detector
distances of1 m (to cover a scattering range of 0.04
-
Figure 4. SDS–PAGE analysis (NOVEXTM, Tris/Gly-cine 4–20%) of
human g1 Fab’ and associated PEGylatedconjugates under nonreducing
and reducing conditions.Lane 1, molecular weight marker
(Mark12TM,NOVEXTM); (Lane 2) human g1 Fab’; (Lane 3)
hFab’-(Br)PEG2� 20k; (Lane 4) DFM-(Br)PEG2� 20k. Leftpanel,
nonreducing conditions; (right panel) reducingcondition.
Figure 5. SDS–PAGE (Tris-Glycine 4–20%) analysisof murine g1
Fab’ and its associated PEG conjugatesunder nonreducing and
reducing conditions. Lane 1,molecular weight markers (Mark 12);
(Lane 2) humang1 Fab’; (Lane 3) murine g1 Fab’; (Lane 4)
mFab’-(L)PEG2� 20k
SOLUTION CONFORMATION OF ANTIBODY FRAGMENTS 2069
in Lane 2 was seen to contain some F(ab’)2evidenced by a band at
approximately 100 kDa:there was also evidence for some free
heavyand light chains at approximately 25 kDa. Theassociated
PEGylated components in Lanes 3 and4 are shown to be >95% pure.
The contamina-tion of the human g1 Fab’ has no consequencesin terms
of interpretation of sedimentationcoefficients as the other
components resolve awaybut renders opaque to interpretation the
SAXSrecords. By contrast, both the murine g1 Fab’ andmFab’-(L)PEG2�
20k were >99% pure (Fig. 5).The mobility of the mFab’-(L)PEG2�
20k (Lane 4)was equivalent in both nonreducing and
reducingconditions confirming that there was no disul-phide bridge
between the heavy and light chainsand that a 20 kDa linear PEG
chain was attachedto the C terminal end of both the heavy and
lightchain.
The results of GF–HPLC analysis for bothhuman g1 Fab’ and murine
g1 Fab’ withassociated PEGylated products can be seen inFigures 6
and 7. GF–HPLC analysis of human g1
DOI 10.1002/jps J
Fab’ (Fig. 6), confirmed the presence of F(ab’)2(28%). The
PEGylated species, hFab’-(Br)PEG2�20k, DFM-(Br)PEG2� 20k and
hFab’-(L)PEG25 k(not shown) were shown to be>98% pure. For
themurine g1 Fab’ and mFab’-(L)PEG2� 20k (Fig. 7),both species were
shown to be >99% pure.
Hydrodynamic Behaviour of PEGs
Figure 8 shows an example of the g�(s) distribu-tion for the
branched 40 kDa PEG plotted withrespect to s (Fig. 8a) and log
es
2 (Fig. 8b), where thelatter takes into account we are dealing
with aquasi-continuous log-normal distribution of mole-cular
weights for the PEG.
The conformation of the PEG chain in aqueoussolution is still a
matter of debate. X-raystructural analysis has shown that
crystallinePEG chains can adopt two extreme
structuralconformations: a zigzag, random coil structure forshorter
chains, or a winding, helical structure forlonger chains.49 It has
been argued from earlierstudies that viscosity50–53 and diffusion
coef-ficient data50 support a random coil conformation,
OURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 6, JUNE 2008
-
Figure 6. GF–HPLC analysis of (a) human g1 Fab’; (b)
hFab’-(Br)PEG2� 20k;(c) DFM-(Br)PEG2� 20k. Zorbax GF450/GF250 HPLC
columns in series with isocraticelution in 0.2 M sodium phosphate,
pH 7.0/10% ethanol at 1.0 mL min�1, detected at awavelength of 280
nm.
2070 LU ET AL.
whereas it has been claimed from calorimetricdata54 that it
adopts a helical conformation. Basedon volumetric studies, Lepori
and Mollica32 favoura compromise model for PEG in solution is
ahydrated flexible coiled polymer with some helicalsegments.
In many cases with high polymeric solutes thereis a unique
relation between the sedimentationcoefficient s and the molecular
weight M. Forexample the extremes are as follows (see, e.g.Ref.
55):
Compact sphere : s � M0:667 (3)
Rigid rod : s � M0:15 (4)
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 6, JUNE
2008
For a flexible coil polymer, s and M follows therelation
that55
s � M0:4�0:5 (5)
or to an approximation
s2 � M (6)
In all three cases loge(s) is the appropriateabscissa. Therefore
if the PEG moiety investi-gated in this study is approximately a
randomcoil conformation, and contains a continuous log-normal
distribution of polymer chain lengths,56
we would expect the g�(s) distribution plot on alog(s2) scale
(or 2 log(s)) which in turn mirrors the
DOI 10.1002/jps
-
Figure 7. GF–HPLC analysis of (a) murine g1 Fab’ and (b)
mFab’-(L)PEG2� 20k.Zorbax GF450/GF250 HPLC columns in series;
isocratic elution in 0.2 M sodiumphosphate, pH 7.0/10% ethanol at
1.0 mL min�1; detected at a wavelength of 280 nm.
SOLUTION CONFORMATION OF ANTIBODY FRAGMENTS 2071
normalised molecular weight distribution to be aunimodal
Gaussian distribution. This unimodalGaussian distribution feature
(see, e.g. Fig. 8b) isclearly evident within the range of
concentrationsstudied (from 0.6 to 2.3 mg/mL). The slightdeviation
could due to the diffusive effects.
The g�(s) distribution yields a modal sedimenta-tion
coefficient. For both PEG reagents, con-siderable nonideality
effects were observed asthe sedimentation coefficient showed a
markeddecrease with increasing concentrations (data notshown). The
concentration-dependence coefficientks estimated from the
extrapolation of 1/s versusconc. plot to zero concentration yields
a value of(81� 6) mL/g using the relation
1
s¼ 1
s0þ ks
s0c (7)
This high ks value found for the 40 kDa PEG ismuch larger than
for globular proteins whichconfirms the flexible expanded
conformation of the
DOI 10.1002/jps J
molecule. Extrapolations of the 1/s versus conc.plot to zero
concentration yields an s020;w value of(0.64� 0.01)S for the linear
20 kDa PEG reagentand (0.82� 0.01)S for the branched 40 kDa
PEGreagent. Interestingly, it is possible to estimatethe molecular
weight M based on the s020;w and ks(after correction for radial
dilution) measure-ments:37 for the 40 kDa PEG reagent, a value ofM
�39000 g/mol is returned.
The sedimentation coefficient is inversely pro-portional to the
frictional ratio following therelation:
s020;w ¼Mwð1 � nr0Þ
6pNAh0
4pNA3nMw
� �1=3 1f=f0
(8)
where Mw is the weight averaged molecularweight (g/mol), n is
the partial specific volume(mL/g), NA is Avogadro’s number
(6.02205� 1023mol�1), r0 (g/mL) and h0 (Poise) are the densityand
viscosity of water at 20.08C respectively.
OURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 6, JUNE 2008
-
Figure 9. The ls-g�(s) distribution for a 1.2
mg/mLhFab’-(Br)PEG2� 20k solution. (a): g�(s) versus s
dis-tribution. (b): log-normal distribution. In (b) the � line
isthe experimental distribution and the red solid lineshows a
single Gaussian curve.
Figure 8. ls-g�(s) distribution of a 0.57 mg/mL 40 kDaPEG
solution. (a) plotted versus s; (b) plotted versuslog(s2), Green:
experimental g�(s) distribution; red:simulated data from
SEDFIT35,36 and black solid linesshows a single Gaussian curve.
Simulated data generat-ed from SEDFIT is based on a
single-component system(using Mw¼ 40 kDa and s¼ 0.8S), showing the
broad-ening of the peak is due to diffusion alone.
2072 LU ET AL.
Following this relation, high frictional ratios(ratio of the
actual friction experienced by aparticle moving through a fluid to
the frictionwhich it would experience if it were a compactsphere of
the same mass)57 were found for the20 kDa PEG ( f/fo �2.5) and the
40 kDa PEG( f/fo �3.0) using the algorithm UNIVERSAL_PARAMS.58
Hydrodynamic Behaviour of PEGylated Conjugates
Interestingly, sedimentation velocity analyses ofthe PEGylated
antibody solutions reveal theirhomogeneous nature. Moreover, the
g�(s) dis-
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 6, JUNE
2008
tribution of these PEGylated antibodies mirrorsthe effect seen
in the analysis of PEG that thedistribution is log-normal with
respect to s or s2
(an example see Fig. 9). This suggests that thehydrodynamic
behaviour of PEGylated antibodyis predominantly dictated by the
attached PEGmoiety. Again, similar to the 40 kDa PEG, themodal
sedimentation coefficients obtained fromg�(s) distribution at
different concentrations wereextrapolated to zero concentration to
eliminatethe effect of nonideality, and the s020;w values
arereported in Table 2.
The weight-average molecular weights ofthe PEGylated conjugates
from sedimentationequilibrium are also shown in Table 3.
Despite
DOI 10.1002/jps
-
Table 2. Experimental Sedimentation and X-Ray Scattering Data
for the Antibody Fragments and their PEGylatedConjugates
Sample s020;w (S) f/fo Rg (Å) Dmax (Å)
Murine g1 Fab’ 3.67� 0.02 1.30 26.4� 1.0 76� 2Human g1 Fab’
3.95� 0.01 1.23 — —Human g1 F(ab’)2 5.49� 0.01 1.40 41.0� 1.0 117�
3mFab-(L)PEG2� 20k 2.12� 0.02 2.75 29.5� 1.0 94� 2hFab’-(L)PEG25 k
2.40� 0.01 2.29 31.2� 1.0 96� 2hFab’-(Br)PEG2� 20k 2.16� 0.02 2.68
— —DFM-(Br)PEG2� 20k 3.41� 0.03 2.47 49.6� 1.0 145� 3
s020;w: sedimentation coefficient; Rg: radius of gyration, Dmax:
maximum dimension of the scattering particle.
SOLUTION CONFORMATION OF ANTIBODY FRAGMENTS 2073
the polydisperse nature of the PEG reactants, themeasured
molecular weight and the theoreticalvalues for the PEGylated
molecule are in reason-able agreement.
Solution Conformation of Antibody Fragmentsand PEGylated
Conjugates
Sedimentation Coefficients
The sedimentation coefficient ðs020;wÞ is a mani-festation of
the overall size and shape of amacromolecule in solution and the
data fromour experiments are summarised in Table 2. Thevalue for
human g1 Fab’ (3.95� 0.01)S is similarto previous determinations.59
With its short hingeremoved, murine g1 Fab’ sediments at the
samerate (3.67� 0.02)S as murine classic g2a Fab’(3.68� 0.01)S.60
Comparison of these last twovalues indicate that the existence of
the hinge maynot alter the overall conformation of the
antibodyfragment and the difference in the hydrodynamicbehaviour
between human g1 Fab’ and murine g1Fab’ is ascribed to the
different antibody speciesused. It is worth pointing out that the
murine g1Fab’ has no upper or middle hinge region since astop codon
was inserted in the gene after the CYS
Table 3. Molecular Weights of the PEGylatedConjugates
SampleTheoretical
M (Da) Mw (Da)
mFab-(L)PEG2� 20k 87200 78000� 3000hFab’-(Br)PEG2� 20k 88100
82000� 4000hFab’-(L)PEG25 k 73100 73000� 2000DFM-(Br)PEG2� 20k
136100 125000� 4000
Mw: experimental (weight-average) molecular weight
fromsedimentation equilibrium.
DOI 10.1002/jps J
residue in CH1. Interestingly, human g1 F(ab’)2has an identical
sedimentation coefficient (withinexperimental error) to the DFM
(covalently linkeddiFab’-maleimide) of (5.53� 0.04)S (Harding,
S.E.and Rhind, S.K. unpublished work).
Despite a large increase in molecular weightdue to the
conjugation of either linear 25 kDaor branched 40 kDa PEG moieties
with theproteins, both hFab’-(L)PEG25k and hFab’-(Br)PEG2� 20k
sediment at a slower rate com-pared to the parent antibody human g1
Fab’ (seeTable 2). Similar sedimentation behaviour hasbeen observed
for the mFab’-(L)PEG2� 20k andDFM-(Br)PEG2� 20k. These data suggest
a sig-nificant effect of PEG on the conformation of allthe antibody
fragments studied, with the increasein frictional ratio f/fo (from
�1.3 to 2.3–2.8):the solution hydrodynamic properties of
theconjugates are clearly dominated by the PEGmoiety ( f/fo
�3.0).
Unfortunately quantifying further the effect onconformation is
rendered difficult because ofthe effects of addition of the PEG on
the waterassociation or time averaged ‘‘hydration’’ of
theantibodies—which also increases the frictionalratio. The large
hydrodynamic size or volume ofPEGylated antibodies in solution has
alreadybeen reported.10
Small Angle X-Ray Scattering
Although the complexities of hydration andflexibility render it
difficult to comment on thedetailed conformation of the protein-PEG
con-jugates it is nonetheless possible to providelimited but
nonetheless useful information onthe conformation of the protein
moiety andthe location of the PEGylation site within thecomplex,
using SAXS to extend the sedimentationvelocity data.
OURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 6, JUNE 2008
-
Figure 10. Superimposition of the experimental p(r)functions
(with error bars) of murine g1 Fab’ (- - -) andmFab-(L)PEG2� 20k
(—). Both molecules have thesame peak M at �30Å. The maximum
dimensions Dmaxare 76 and 95 Å respectively.
Figure 11. Superimposition of the experimental p(r)functions
(with error bars) of human g1 F(ab’)2 (- - -) andDFM-(Br)PEG2� 20k
(—). For human g1 F(ab’)2, peaksM1 and M2 occur at �32 Å and �75
Å respectively, withDmax �120 Å. For DFM-(Br)PEG2� 20k peaks M1
andM2 occur at rM1 �35 Å and rM2�90 Å respectively, withDmax �145
Å.
2074 LU ET AL.
Small angle SAXS measures the relativescattering intensity
between the scattering par-ticle and the solvent.29 The Guinier
approxima-tion42 of the scattering profile yields the radiusof
gyration (Rg) of the particle in solution andindicates its
elongation or compactness. Fouriertransformation of the scattering
profile gives thedistance distribution function p(r) as a function
ofscattering distance r. This is characterised by oneor maxima (M)
and the maximum dimension ofthe scattering particle (Dmax) both of
which arecharacteristic features of the particle in solution:the
algorithm in GNOM41 allows to calculate p(r)as well as a further
estimate for Rg (encouraginglyRg values from this procedure which
wereconsistent with those from the Guinier analysis).
Using these procedures a radius of gyration of26.4 Å was found
for the murine g1 Fab’, similar tothe previous determination.61
Comparing thePEGylated conjugates with their parent anti-bodies
(Table 2), scattering data have shown thatthe effect of PEGylation
is to contribute to a more‘‘extended’’ conformation in solution as
indicatedby the higher Rg and Dmax values. By attachingtwo linear
20 kDa PEG to the C-termini of theheavy and light chains of the
murine g1 Fab’, theresulting conjugate mFab’-(L)PEG2� 20k has aDmax
of 94 Å, suggesting a �20 Å increasecompared to the murine g1
Fab’. The radius ofgyration data are consistent with the
Dmaximplying that the PEGylated molecule is moreelongated. More
pronounced differences werefound between the human g1 F(ab’)2 and
DFM-(Br)PEG2� 20k, with a �9 Å increase in Rg and a�30 Å increase
in Dmax.
The distance distribution function p(r) of ascattering particle
reflects the shape and massdistribution of the molecule.29,62
Figures 10 and11 show the superimpositions of the p(r) functionof
the antibody fragments and their PEGylatedconjugates. The p(r)
function represents thedistribution of all intra-particle
scattering vectorsand therefore the maximum M in p(r) correspondsto
the most frequently occurring interatomic dis-tance within the
molecular structure.29,63 Murineg1 Fab’ and mFab’-(L)PEG2� 20k both
have thismaximum M at a value of r of �30 Å (Fig. 10): thisvalue
refers to the most commonly occurringdistance between the
scattering centres in a singleFab’. Thus we can conclude that no
significantconformational change to the antibody fragmentoccurs
upon PEGylation, that is, the structure ofthe Fab’ fragment itself
remains essentiallyunaltered. Parts of the PEGylated molecule
extend
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 6, JUNE
2008
over larger distances (e.g. between the PEG moietyand the
protein) and therefore contribute to thedistribution of longer
distances in the p(r) functionwhich result in an increase in Rg and
Dmaxcompared to the isolated Fab’ fragment.
Similarly, there is little tendency for the Fab’domain within
DFM-(Br)PEG2� 20k to undergoconsiderable conformation changes when
com-pared to the human g1 F(ab’)2. Analogous posi-tions of the
first maxima M1 are found at (32–35)Å (Fig. 11) that can be
assigned to the mostcommonly occurring distance within a single
Fab’.The second maximum peak M2 describes the mostcommon distance
within the whole molecule andtherefore can be ascribed to the
distance between
DOI 10.1002/jps
-
SOLUTION CONFORMATION OF ANTIBODY FRAGMENTS 2075
the two Fab’s. As a bis-maleimide linker wasused for the
synthesis of DFM-(Br)PEG2� 20k,an increase of 15 Å in peak M2 of
DFM-(Br)PEG2� 20k (90 Å) compared to the humang1 F(ab’)2 (75 Å)
illustrates the conformationalvariation between these two molecules
due to theadduct of the bis-maleimide derivative of thebranched
PEG.
Figure 13. Superimposition of experimental p(r)function (with
error bars) of the murine g1 Fab’ withp(r) functions of two Fab’
crystallographic models cal-culated from HYDROPRO46: 1UCB (— ��—
��) and 1BBJ( ). For the murine g1 Fab’, the maximum M occursat �30
Å and the maximum dimensions Dmax occurs at�76 Å
respectively.
DISCUSSION
Ab Initio Modelling
Ab initio shape reconstruction using the programDAMMIN43 was
carried out to visualise theconformations of the antibodies and
their PEGy-lated conjugates in solution For each molecule,10 low
resolution models obtained from differentDAMMIN runs were analysed
and the stableconstructions (NSD< 0.8) were averaged thatyielded
a model representing the most probablefeatures of the solution (see
Materials andMethods Section).
To examine the validity of the shape reconstruc-tion, the ab
initio model of the murine g1 Fab’ wascompared with the
crystallographic models of twoFab’ molecules (accession code 1UCB
and 1BBJrespectively). Superimpositions of the ab initiomodel of
murine g1 Fab’ with the crystallographicmodels using SUPCOMB47 and
MASSHA48 are invery good agreement with all three Fab’ modelsand
have a similar volume (an example seeFig. 12). The scattering
properties of the crystal-
Figure 12. Superimposition of the Fab’ crystal struc-ture (PDB
accession code 1UCB, red cartoon) with theab initio model of murine
g1 Fab’ (grey spheres). Thecysteine in the light chain at position
214 is highlightedby green spheres.
DOI 10.1002/jps J
lographic models of two Fab’ molecules (accessioncode 1UCB and
1BBJ respectively) were calculat-ed using CRYSOL45 and HYDROPRO46
(data notshown). Superimposition of the p(r) functions (seeFig. 13)
for the three different Fab’ models yieldsimilar positions for the
maxima M and Dmax andreflect the excellent harmony revealed
betweenab initio models and crystal structures (Fig. 12).
Correspondingly, ab initio models of mFab-(L)PEG2� 20k and
hFab’-(L)PEG25k were calcu-lated and superimposed with the model
ofthe murine g1 Fab’ (see Figs. 14 and 15).Distinct structural
features are revealed in the
Figure 14. Superimposition of ab initio models ofmFab-(L)PEG2�
20k (transparent cyan spheres) withmurine g1 Fab’ (transparent grey
spheres). For orienta-tion the red ribbon model represents the Fab’
crystalstructure as shown in Figure 9.
OURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 6, JUNE 2008
-
Figure 15. Superimposition of ab initio models ofhFab’-(L)PEG25
k (transparent pink spheres) withmurine g1 Fab’ (transparent grey
spheres). For orienta-tion the red ribbon model represents the Fab’
crystalstructure in analogy to Figures 9 and 11. Figures 9,11and 12
were produced with the routine PYMOL.65
2076 LU ET AL.
superimpositions of the Fab’ with PEGylationconjugates and
clearly demonstrates that theFab’ is essentially unchanged by
PEGylation. Theextension in the PEGylated model is attributed tothe
conjugation of PEG. However, it is apparentthat the increase in
volume of the PEGylatedmodel is smaller than expected considering
thesize of the PEG moiety. With the significantincrease in
frictional ratio as observed by sedi-mentation velocity, it might
be anticipatedthat the PEGylated molecule being much
more‘‘extended’’ or occupying a larger excluded volume.An
explanation can be found in the fact that thePEG molecules are
highly dynamic and flexible insolution and primarily the immediate
interactionregions with the antibody will contribute to astrong
scattering contrast between solvent andmacromolecule. Besides, the
shape restorationprocedure with DAMMIN works more reliablyfor
compact and rigid molecular configurationsand it may be less
sensitive to loosely boundand dynamically disordered structural
arrange-ments.64 Moreover, as the electron density withina
PEGylated molecule is not homogeneous and ischaracterised by high
solvation, the scatteringcontrast between the PEG moiety and the
solventis smaller compared to the one between proteinand
solvent.
This deviation from the expected particlevolume suggests that
the ab initio restorationdoes not represent fully the effective
volume of
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 6, JUNE
2008
the whole PEGylated molecule. Considering theabove reasons it
could be inferred that only themore ordered and rigid parts of the
PEG moietieshave been manifested in the shape
calculations:contributions on the other hand from the dyna-mically
disordered PEG segments had a contrastfor X-rays that were only
marginally higher thanthat for the buffer solution. Further
studiesinvolving contrast variation small angle neutronscattering
(SANS) may help to resolve the issue onthe conformation and
contribution of the PEGmoiety. Although SAXS fails to reveal
detailsabout the conformation of the whole PEG compo-nent,
nonetheless it is still possible to commenton the way PEG is bound
to the protein. Forinstance, models of mFab’-(L)PEG2� 20k
andhFab’-(L)PEG25k have shown that the attachedPEG moiety extends
from one end of the Fab’. Asuperimposition of the crystallographic
modelswith the ab initio model of hFab’-(L)PEG25k isachievable so
that the PEG moiety is attached tothe Fab’ fragment through the
free cysteine thatis exposed at the C-termini of the Fab’
molecule(see Fig. 9). This is consistent with the
conjugationchemistry applied in site-specific PEGylation(Fig.
2).
Concluding Remarks
AUC and measurement of the translational fric-tional ratio has
shown that for all PEGylated-antibody fragment complexes studied,
the hydro-dynamic properties are dominated by the PEGmoiety. SAXS
at low values of r (distance betweenscattering centres) as
manifested by the rM and Rgvalues, indicate that the conformation
of Fab’ itselfis essentially unchanged by incorporation into
thecomplex. The globular Fab’ therefore appears to‘‘sit’’ inside
the flexible PEG: although attachedat a specific point, because of
its much greaterflexibility the time-averaged flexible
conformationof the PEG may effectively cover part or all ofthe
Fab’.
ACKNOWLEDGMENTS
We thank the United Kingdom Engineering andPhysical Science
Research Council for supportingthis work, and the Council for the
Central Labora-tory of the Research Councils for access to the
SRS,Daresbury. We thank Alistair Henry at UCB-Cell-tech (Slough,
UK) for useful discussions, Helen
DOI 10.1002/jps
-
SOLUTION CONFORMATION OF ANTIBODY FRAGMENTS 2077
Brand and Gayle Phillips, also at UCB-Celltech(Slough, UK) for
help in the purification and PEGy-lation of the Fab’ fragments and
Dr. Gordon Morris(NCMH) for the SEC-MALLs check on our mole-cular
weights.
REFERENCES
1. Holliger P, Hudson PJ. 2005. Engineered antibodyfragments and
the rise of single domains. Nat Bio-technol 23:1126–1136.
2. King DJ, Adair JR, Angal S, Low DC, Proudfoot KA,Lloyd JC,
Bodmer M, Yarranton GT. 1992. Ex-pression, purifucation and
characterisation of amouse:human chimeric antibody and chimericFab’
fragment. Biochem J 281:317–323.
3. Sun CZ, Wirsching P, Janda KD. 2003. EnablingScFv as
multi-drug carriers: A dendritic approach.Bioorg Med Chem
11:1761–1768.
4. Skerra A, Pluckthun A. 1988. Assembly of a func-tional
immunoglobulin Fv fragment in Escherichiacoli. Science
240:1038–1041.
5. Carter P, Kelley RF, Rodrigues ML, Snedecor B,Covarrubias M,
Velligan MD, Wong WLT, RowlandAM, Kotts CE, Carver ME, Yang M,
Bourell JH,Shepard HM, Henner D. 1992. High level Escher-ichia coli
expression and production of a bivalenthumanized antibody fragment.
Bio/Technology10:163–167.
6. King DJ, Turner A, Farnsworth AP, Adair JR,Owens RJ, Pedley
RB, Baldock D, Proudfoot KA,Lawson AD, Beeley NR. 1994. Improved
tumortargeting with chemically cross-linked recom-binant antibody
fragments. Cancer Res 54:6176–6185.
7. Casey JL, King DJ, Chaplin LC, Haines AM, PedleyRB, Mountain
A, Yarranton GT, Begent RH. 1996.Preparation, characterisation and
tumour target-ing of cross-linked divalent and trivalent
anti-tumour Fab’ fragments. Br J Cancer 74:1397–1405.
8. Chen J, Jaracz S, Zhao X, Chen S, Ojima I.
2005.Antibody-cytotoxic agent conjugates for cancertherapy. Expert
Opin Drug Deliv 2:873–890.
9. Chapman AP, Antoniw P, Spitali M, West S,Stephens S, King DJ.
1999. Therapeutic antibodyfragments with prolonged in vivo
half-lifes. NatBiotechnol 17:780–783.
10. Chapman AP. 2002. PEGylated antibodies andantibody fragments
for improved therapy: A review.Adv Drug Deliver Rev 54:531–545.
11. Koumenis IL, Shahrokh Z, Leong S, Hsei V, DeforgeL, Zapata
G. 2000. Modulating pharmacokinetics ofan anti-interleukin-8
F(ab’)2 by amine-specificPEGylation with preserved bioactivity. Int
J Pharm198:83–95.
DOI 10.1002/jps J
12. Tsutsumi Y, Onda M, Nagata S, Lee B, KreitmanRJ, Pastan I.
2000. Site-specific chemical modi-fication with polyethylene glycol
of recombinantimmunotoxin anti-Tac(Fv)-PE38 (LMB-2)
improvesantitumor activity and reduces animal toxicity
andimmunogenicity. PNAS 97:8548–8553.
13. Delgado C, Pedley RB, Herraez A, Boden R, BodenJA, Keep PA,
Chester KA, Fisher D, Begent RHJ,Francis GE. 1996. Enhanced tumour
specificity ofan anti-carcinoembrionic antigen Fab’ fragmentby
poly(ethylene glycol) (PEG) modification. Br JCancer
73:175–182.
14. Bailon P, Berthold W. 1998. Polyethylene glycol—conjugated
pharmaceutical proteins. PharmaceutSci Technol Today 1:352–356.
15. Harris JM, Chess RB. 2003. Effect of PEGylation
onpharmaceuticals. Nat Rev Drug Discov 2:214–221.
16. Harris JM. 1992. Introduction to biotechnical andbiomedical
applications of poly(ethylent glycol). In:Harris JM, editor.
Poly(ethylene glycol) chemistry:Biotechnical and biomedical
applications. PlenumPress. New York: pp 1–29.
17. Zalipsky S, Harris JM. 1997. Introduction tochemistry and
biological applications of poly(ethy-lene glycol). In: Harris JM,
Zalipsky S, editors.Poly(ethylene glycol) chemistry and
biologicalapplications. Washington, DC: American ChemicalSociety.
pp 1–5.
18. Roberts MJ, Bentley MD, Harris JM. 2002. Chem-istry for
peptide and protein PEGylation. Adv DrugDeliver Rev 54:459–476.
19. Wang Y, Youngster S, Grace M, Bausch J, BordensR, Wyss DF.
2002. Structural and biological char-acterization of pegylated
recombinant interferonalpha-2b and its therapeutic implications.
AdvDrug Deliver Rev 54:547–570.
20. Fee CJ, Van Alstine JM. 2006. PEG-proteins: Reac-tion
engineering and separation issues. Chem EngSci 61:924–939.
21. Fee CJ, Van Alstine JM. 2004. Prediction of theviscosity
radius and the size exclusion chromato-graphy behavior of PEGylated
proteins. Bioconju-gate Chem 15:1304–1313.
22. Kodera Y, Matsushima A, Hiroto M, Nishimura H,Ishii A, Ueno
T, Inada Y. 1998. PEGylaion of pro-tiens and bioactive substances
for medical andtechnical applications. Prog Polym Sci
23:1233–1271.
23. Rajender Reddy K, Modi MW, Pedder S. 2002. Useof
peginterferon alfa-2a (40 KD) (Pegasys1) for thetreatment of
hepatitis C. Adv Drug Deliver Rev54:571–586.
24. Charles SA, Harris JM, Pedder S, Kumar S. 2000.Improving
hepatitis C therapy: Attaching a poly-ethylene glycol ‘‘tail’’ to
interferon for better clinicalproperties. Mod Drug Discov
5:59–67.
25. Monfardini C, Schiavon O, Caliceti P, MorpurgoM, Harris JM,
Veronese FM. 1995. A branced
OURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 6, JUNE 2008
-
2078 LU ET AL.
monomethoxypoly(ethelene glycol) for protein mod-ification.
Bioconjugate Chem 6:62–69.
26. Veronese FM, Harris JM. 2002. Introduction andoverview of
peptide and protein pegylation. AdvDrug Deliver Rev 54:453–456.
27. In: Scott D, Harding SE, Rowe AJ, editors. 2005.Analytical
ultracentrifugation: Techniques andmethods. Cambridge: Royal
Society of Chemistry.
28. In: Harding SE, Rowe AJ, Horton JC, editors.1992. Analytical
ultracentrifugation in biochemis-try and polymer science.
Cambridge: Royal Societyof Chemistry. 629.
29. Glatter O, Kratky O. 1982. Small angle X-ray scat-tering.
London: Academic Press.
30. Koch MHJ, Vachette P, Svergun DI. 2003.Small-angle
scattering: A view on the properties,structures and structural
changes of biologicalmacromolecules in solution. Q Rev Biophys
36:147–227.
31. Laue TM, Shah BD, Ridgeway TM, Pelletier SL.1992.
Computer-aided interpretation of analyticalsedimentation data for
proteins. In: Harding SE,Rowe AJ, Horton JC, editors. Analytical
ultracen-trifugation in biochemistry and polymer science.Royal
Society of Chemistry. Cambridge: pp 90–125.
32. Lepori L, Mollica V. 1978. Volumetric properties ofdilute
aqueous solutions of poly(ethylene glycols).J Polym Sci
16:1123–1134.
33. Nichol LW, Ogston AG, Wills PR. 1981. Effect ofinert
polymers on protein self-association. FEBSLett 126:18–20.
34. Durchschlag H, Zipper P. 2005. Calculation ofvolume,
surface, and hydration properties of biopo-lymers. In: Scott D,
Harding SE, Rowe AJ, editors.Analytical ultracentrifugation:
Techniques andmethods. Royal Society of Chemistry. Cambridge:pp
389–431.
35. Schuck P. 2000. Size distribution analysis of
macro-molecules by sedimentation velocity ultracentrifu-gation and
Lamm equation modeling. Biophys J78:1606–1619.
36. Dam J, Schuck P. 2004. Calculating sedimentationcoefficient
distributions by direct modeling of sedi-mentation velocity
concentration profiles. MethodsEnzymol 384:185–212.
37. Rowe AJ. 1992. The concentration dependence ofsedimentation.
In: Harding SE, Rowe AJ, HortonJC, editors. Analytical
ultracentrifugation in bio-chemistry and polymer science. Royal
Society ofChemistry. Cambridge: pp 394–406.
38. Cölfen H, Harding SE. 1997. MSTARA andMSTARI: Interactive
PC algorithms for simple,model independent evaluation of
sedimentationequilibrium data. Eur Biophys J 25:333–346.
39. Creeth JM, Harding SE. 1982. Some observationson a new type
of point average molecular weight.J Biochem Biophys Methods
7:25–34.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 6, JUNE
2008
40. Boulin CJ, Kempf R, Gabriel A, Koch MHJ. 1988.Data
acquisition systems for linear and area x-raydetectors using
delay-line readout. Nucl Instr MethPhys Res 269:312–320.
41. Semenyuk AV, Svergun DI. 1991. GNOM-a pro-gram package for
small-angle scattering data pro-cessing. J Appl Crystal
24:537–540.
42. Guinier A, Fournet G. 1995. Small angle scatteringof X-rays.
Wiley: New York.
43. Svergun DI. 1999. Restoring low resolution struc-ture of
biological macromolecules from solutionscattering using simulated
annealing. Biophys J76:2879–2886.
44. Volkov VV, Svergun DI. 2003. Uniqueness ofab initio shape
determination in small-angle scat-tering. J Appl Cryst
36:860–864.
45. Svergun DI, Barberato C, Koch MHJ. 1995. CRY-SOL-a program
to evaluate x-ray solution scatter-ing of biological macromolecules
from atomiccoordinates. J Appl Cryst 28:768–773.
46. Garcia de la Torre J, Huertas ML, Carrasco B.
2000.Calculation of hydrodynamic properties of globularproteins
from their atomic-level structure.Biophys J 78:719–730.
47. Kozin MB, Svergun DI. 2000. Automated matchingof high- and
low-resolution structural models.J Appl Cryst 34:33–41.
48. Konarev PV, Petoukhov MV, Svergun DI. 2001.MASSHA-a graphics
system for rigid-bodymodelling of macromolecular complexes
againstsolution scattering data. J Appl Cryst 34:527–532.
49. Heymann B, Grubmuller H. 1999. Elastic proper-ties of
poly(ethylene-glycol) studied by moleculardynamics stretching
simulations. Chem Phys Lett307:425–432.
50. Chew B, Couper A. 1976. Diffusion, Viscosity
andsedimentation of poly(ethylene oxide) in water.J Chem Soc,
Faraday Trans 72:382.
51. Branca C, Magazu S, Maisano G, Migliardo P,Migliardo F,
Romeo G. 2002. Hydration parametersof aqueous solutions of
poly(ethylene glycol)s byviscosity data. Physica Scripta 66:175–
179.
52. Bailey FE, Callard RW. 1959. Some properties ofpoly(ethylene
oxide) in aqueous solution. J ApplPolym Sci 1:56–62.
53. Branca C, Magazu S, Maisano G, Migliardo F,Migliardo P,
Romeo G. 2003. Study of conforma-tional properties of poly(ehtylene
oxide) by SANSand PCS techniques. Physica Scripta 67:551–551.
54. Maron SH, Filisko FE. 1972. Heats of solution anddilution
for poly(ethylene oxide) in several solvents.J Macromol Sci Phys
6:79–90.
55. Smidrsød O, Andresen IL. 1979. Biopolymerkjemi.Trondheim,
Norway: Tapir Press.
56. Fujita H. 1962. Mathematical theory of sedimenta-tion
analysis. Academic Press. New York: 315.
DOI 10.1002/jps
-
SOLUTION CONFORMATION OF ANTIBODY FRAGMENTS 2079
57. van Holde KE. 1985. Chapter 5: Sedimentation.Physical
Biochemistry. Prentice-Hall, Inc. NewJersey: pp 110–133.
58. Harding SE, Cölfen H, Aziz Z. 2005. The ELLIPSsuite of
whole-body protein conformation algo-rithms for Microsoft Windows.
In: Scott D, HardingSE, Rowe AJ, editors. Analytical
ultracentrifuga-tion techniques and methods. Royal Society
ofChemistry. Cambridge: pp 468–483.
59. Carrasco B, Garciade la Torre J, Byron O, King D,Walters C,
Jones S, Harding SE. 1999. Novel size-independent modeling of the
dilute solution confor-mation of the immunoglobulin IgG Fab’
domainusing SOLPRO and ELLIPS. Biophys J 77:2902–2910.
60. Lu Y. 2007. PhD Thesis: Solution conformation ofengineered
antibodies. School of Biosciences, ed.,Nottingham: University of
Nottingham
61. Morgan PJ, Byron OD, Harding SE. 1992. Thesolution
conformation of novel antibody fragmentsstudied using analytical
ultracentrifugation.
DOI 10.1002/jps J
Discovery 1–4. (Beckman Instruments, Palo Alto,USA) DS-834.
62. Svergun DI. 1992. Determination of the re-gularisation
parameter in indirect transformusing perceptual criteria. J Appl
Crystal 25:495–503.
63. Furtado PB, Whitty PW, Robertson A, Eaton JT,Almogren A,
Kerr MA, Woof JM, Perkins SJ. 2004.Solution structure determination
of monomerichuman IgA2 by X-ray and neutron scattering, ana-lytical
ultracentrifugation and constrained model-ling: A comparison with
monomeric human Ig A1.J Mol Biol 338:921–941.
64. Zipper P, Durchschlag H, Krebs A. 2005. Model-ling of
Biopolymers. In: Scott D, Harding SE, RoweAJ, editors. Analytical
ultracentrifugation: Techni-ques and methods. Royal Society of
Chemistry.Cambridge: pp 320–371.
65. DeLano WL. 2002. The PyMOL Molecular GraphicsSystem. DeLano
Scientific. San Carlos. CA, USA,http://wwwpymolorg.
OURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 6, JUNE 2008