ANALYTICAL
BIOCHEMISTRY
Analytical Biochemistry 351 (2006) 241–253
www.elsevier.com/locate/yabio
High-throughput aYnity ranking of antibodies using surface plasmon
resonance microarrays
Dina Wassaf a, Guannan Kuang a, Kris Kopacz a, Qi-Long Wu a, Que Nguyen a, Mark Toews a,
Janja Cosic a, Judith Jacques a, Steve Wiltshire b,1, Jeremy Lambert c, Csaba C. Pazmany a,
Shannon Hogan a, Robert C. Ladner a, Andrew E. Nixon a, Daniel J. Sexton a,¤
b
a
Dyax Corp., Cambridge, MA 02139, USA
HTS Biosystems, East Hartford, CT 06108, USA
c
PhyNexus, San Jose, CA 95136, USA
Received 9 November 2005
Available online 10 February 2006
Abstract
A method was developed to rapidly identify high-aYnity human antibodies from phage display library selection outputs. It combines
high-throughput Fab fragment expression and puriWcation with surface plasmon resonance (SPR) microarrays to determine kinetic constants (kon and koV) for 96 diVerent Fab fragments in a single experiment. Fabs against human tissue kallikrein 1 (hK1, KLK1 gene product) were discovered by phage display, expressed in Escherichia coli in batches of 96, and puriWed using protein A PhyTip columns.
Kinetic constants were obtained for 191 unique anti-hK1 Fabs using the Flexchip SPR microarray device. The highest aYnity Fabs discovered had dissociation constants of less than 1 nM. The described SPR method was also used to categorize Fabs according to their ability to recognize an apparent active site epitope. The ability to rapidly determine the aYnities of hundreds of antibodies signiWcantly
accelerates the discovery of high-aYnity antibody leads.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Antibody phage display; Serine protease; kallikrein; Antibody inhibitors; Surface plasmon resonance; Antibody arrays
Phage display has been shown to be a powerful discovery tool for diverse Welds such as proteomics [1,2], enzyme
engineering [3], aYnity chromatography [4,5], small-molecule therapeutics [6,7], and the development of fully human
therapeutic antibodies [8,9]. Phage display often has relied
on the use of subsequent aYnity maturation steps to generate antibodies of suYciently high aYnity to serve as therapeutic candidates [10–13]. However, recent advances in
library construction methods have led to the generation of
Fab libraries capable of routinely producing high-aYnity
antibodies (Kd < 10 nM) without the need for time-consum*
Corresponding author. Fax: +1 617 225 2501.
E-mail address: [email protected] (D.J. Sexton).
1
Present address: Charles River Laboratories, Wilmington, MA 01887,
USA.
0003-2697/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2006.01.043
ing aYnity maturation [14]. Furthermore, the introduction
of automated screening methods to the phage display process provides the opportunity to evaluate hundreds of antibodies in downstream assays.
Automated enzyme-linked immunosorbent assay
(ELISA)2 and microarray methods have been shown to be
eVective primary screens for the initial identiWcation of
2
Abbreviations used: ELISA, enzyme-linked immunosorbent assay;
SPR, surface plasmon resonance; hK1 (KLK1 gene product), human tissue
kallikrein 1; YPD, yeast–peptone–dextrose; BMGY, buVered minimal
glycerol complex medium; BMM, buVered minimal methanol medium;
pfu, plaque-forming units; PEG, polyethylene glycol; IPTG, isopropyl-D-1-thiogalactopyranoside; BSA, bovine serum albumin; TMB, 3,3⬘,
5,5⬘-tetramethylbenzidine; PBS, phosphate-buVered saline; ROI, region of
interest; RCU, resonance change units; AMC, 7-amino-4-methylcoumarin; CV, coeYcient of variation.
242
High-throughput aYnity ranking of antibodies / D. Wassaf et al. / Anal. Biochem. 351 (2006) 241–253
phage hits [15–19]. Discovery of the most potent antibodies
identiWed in the primary screen depends on the development and use of suitable secondary screening assays. Secondary assays should minimally provide a relative aYnity
ranking and, if possible, reliable estimates of kinetic or
equilibrium aYnity constants for each of the hits identiWed
in the primary screen. Surface plasmon resonance (SPR)
methods have been shown to be well suited to measure the
kinetics of antibody–antigen interactions [20] and promise
to be eVective secondary screening assays for phage display.
SPR-based biosensors are able to detect, in real time, the
kinetics of association and dissociation as proteins absorb
and desorb from the sensor surface. This is achieved
through measurement of the change in refractive index of
the buVer near the sensor, with the change in refractive
index being proportional to the protein mass bound to the
sensor surface. An SPR secondary screening assay for
phage display must be capable of rapidly analyzing all of
the unique antibodies discovered in the primary screen.
Sequential injection SPR methods have been developed for
antibodies obtained from hybridoma screens that may be
applicable for phage display [21]. In contrast, the Flexchip
SPR instrument uses a highly parallel approach to provide
estimates of kinetic constants for up to 400 diVerent protein–protein interactions in a single experiment [22–24]. The
Flexchip system uses a slide composed of an optical grat
ing that is coated with a thin layer of gold onto which proteins are immobilized in a 1 £ 1-cm microarray. The application of array-based biosensor in drug discovery has the
potential to dramatically increase the throughput and success rate of such programs.
The ability to simultaneously determine the binding
properties of hundreds of candidate therapeutic proteins
shifts the bottleneck in a therapeutic discovery program
from analysis to protein puriWcation. To address this bottleneck, advances in automated protein puriWcation procedures have been reported [25,26]. One technology that has
been introduced is PhyTip columns, which consist of
pipette tips that contain a small amount (5 l) of puriWcation resin, such as protein A–Sepharose, encased in hydrophilic screens at the ends of the tips [25,26]. Antibody or
protein puriWcation using the PhyTip columns can be performed in parallel using laboratory equipment such as multichannel pipettors or using automated workstations
capable of handling 96 PhyTips at once. An advantage of
the technology is the ability to elute the enriched protein in
a small volume, thereby promoting recovery at suYciently
high protein concentrations for further characterization.
Here we describe a procedure that combines automated
Fab puriWcation using protein A PhyTip columns with
high-throughput determination of kinetic constants of
Fabs using the Flexchip SPR technology. We illustrate this
approach using Fabs identiWed from a phage selection campaign against human tissue kallikrein 1 (hK1, KLK1 gene
product). hK1 is a serine protease that is responsible for the
production of Lys-bradykinin, which is a potent mediator
of inXammation [27]. The described method is shown to
serve as an eVective secondary screening assay for the discovery of potent antibodies from phage display libraries
and can greatly reduce the need for time-consuming aYnity
maturation eVorts.
Materials and methods
hK1 expression and puriWcation
The cDNA encoding the KLK1 gene was obtained from
Invitrogen and subcloned into the pPICZ vector (Invitrogen), which was then integrated into the genomic DNA of
Pichia pastoris (X33) according to the supplier’s instructions. The clone expressing the highest amount of hK1 was
identiWed by measuring the hK1 protease activity in the
medium, and that clone was scaled up for protein production. BrieXy, a culture of P. pastoris was grown overnight in
yeast–peptone–dextrose (YPD, 1 ml) at 30 °C with vigorous
shaking, and 2.5 ml of this was used to inoculate 4 L buVered
minimal glycerol complex medium (BMGY), which was
then grown for 48 to 60 h at 30 °C. The cells were collected
by centrifugation (1500g for 5 min), resuspended in 1 L
buVered minimal methanol medium (BMM), and grown at
30 °C for 4 days with daily additions of 1% methanol. The
cells were then removed by centrifugation (9700g for
15 min), and the medium was Wltered through a 0.45-m
Wlter. The medium was extensively diaWltered against 3 L of
50 mM Hepes (4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid, pH 7.8) and concentrated to approximately
150 ml using a 10-kDa molecular weight cutoV membrane
(Amicon). The concentrated medium was stored at 4 °C in
the presence of 10 mM benzamidine to prevent autoproteolysis. Phenyl Sepharose (50 ml) was then added to the concentrated medium and rotated 4 h at room temperature to
remove excess pigment. The phenyl Sepharose was removed
by Wltration through a 0.45-m Wlter before application of
the medium at a rate of 1 ml/min on a Q-Sepharose ion
exchange column (two 1-ml Q-Sepharose Hi-Trap columns
[Amersham] connected in series) equilibrated in buVer A
(20 mM sodium phosphate, pH 8.0). The column was
washed with 10 ml buVer A (containing 200 mM sodium
chloride) and eluted with a linear gradient to buVer B
(20 mM sodium phosphate, 1 M sodium chloride, pH 8.0)
over 60 min at a Xow rate of 1 ml/min. The fractions containing hK1 activity were then loaded onto a benzamidine
Sepharose column (1-ml Hi-Trap column) that was equilibrated in buVer C (50 mM Hepes, 500 mM sodium chloride,
pH 7.8). The benzamidine column was washed with 10 ml
buVer C and eluted with a linear gradient over 60 min at a
Xow rate of 1 ml/min to buVer D (100 mM sodium citrate,
500 mM sodium chloride, pH 3.0). Active fractions were
then pooled, buVer exchanged into storage buVer (50 mM
Hepes, 10 mM benzamidine, pH 7.8), and stored at ¡80 °C.
The above expression and puriWcation procedure yielded
approximately 300 g puriWed hK1. By SDS–PAGE analysis, the purity of the hK1 was greater than 90%, and its identity was conWrmed by tryptic digests and mass spectrometry.
High-throughput aYnity ranking of antibodies / D. Wassaf et al. / Anal. Biochem. 351 (2006) 241–253
Phage display selection strategy 1
The antibody phage display library used here is a phagemid system (known as the Dyax Fab 310 library) that displays antibody Fab fragments fused to the gene III protein
of M13 and contains a diversity of approximately 3.5 £ 1010
diVerent antibodies [14]. Prior to initiating each round of
selection, the antibody library was depleted of streptavidin
binders by incubating the phage library with beads (0.5 mg)
in 0.5 ml PBST (43 mM sodium phosphate, 1.5 mM potassium phosphate, 0.14 M sodium chloride, 2.7 mM potassium chloride, 0.1% Tween 20, pH 7.4) for 5 min before
washing 12 times with PBST using a Dynal magnet. This
streptavidin depletion step was repeated three times. Round
1 was performed by incubating 1 £ 1013 plaque-forming
units (pfu) phage with immobilized hK1 for 5 min in 1 ml
PBST at room temperature. Immobilized hK1 was prepared by capturing 50 nM biotinylated hK1 with 3 mg
streptavidin-coated magnetic beads (cat. no. 112.06, Dynal)
in 300 l PBST. The beads were washed 12 times with PBST
to remove free or weakly associated phage and then were
treated with 25 M aprotinin to elute phage bound to the
active site of hK1. The eluted supernatant and beads were
then ampliWed separately by infecting 20 ml of TG1 cells at
37 °C for 30 min before adding 50 l of 2 £ 1012/ml pfu
M13KO7 helper phage and incubating an additional
30 min at 37 °C. The ampliWcation was completed by adding
80 ml 2XYT media (containing 100 g/ml ampicillin and
50 g/ml kanamycin) and an overnight incubation at 30 °C.
Following ampliWcation, phage were Wltered (cat. no.
431174, Corning) and precipitated with a 60-min incubation (1:5 volume, 40% polyethylene glycol [PEG], 2.5 M
NaCl) on ice. The precipitate was resuspended in 1 ml
PBST and tittered for use in the next round of selection.
Round 2 of selection strategy 1 was performed as
described for round 1 except that the amounts of target
protein and phage both were reduced. For round 2, 1 £ 1011
pfu phage were incubated with hK1 immobilized by incubating 10 nM biotinylated hK1 with 0.5 mg streptavidincoated magnetic beads in 500 l PBST. Round 1 produced
two ampliWed phage inputs (phage eluted with aprotinin
and phage remaining bound to the beads) for round 2,
which then produced four outputs, each of which was
screened by ELISA.
Phage display selection strategy 2
Selection strategy 2 was performed as described for
selection strategy 1 except that three rounds were performed and the phage were treated three times with the
immobilized hK1–aprotinin complex at the beginning of
each round. The immobilized hK1–aprotinin complex was
prepared by incubating 50 nM biotinylated hK1 and 6 M
aprotinin with 2.5 mg streptavidin-coated magnetic beads
and for 30 min at room temperature. Free aprotinin was
removed from the complex by washing the beads Wve times
with PBST. The phage titer decreased from 1 £ 1013 pfu for
243
round 1, to 1 £ 1011 pfu for round 2, to 1 £ 1010 pfu for
round 3. The amount of immobilized hK1 was maintained
constant for each of the three rounds of selection (50 nM
biotinylated hK1 on 2.5 mg streptavidin-coated magnetic
beads in 500 l PBST). In each round of selection, the phage
that were captured on the beads were ampliWed as
described above and used as input for the subsequent
round.
Batch formation of Fab expression vector
After completing the above selection strategies, phage
DNA from each of the Wve diVerent phage outputs was processed separately to remove the gene III stump of M13 and
thereby create an expression vector capable of expressing
soluble Fab fragment. To remove the gene III stump, 1.5 g
double-stranded phage DNA was isolated and digested
with 7.5 U of MluI for 2 h at 37 °C. Digested DNA was then
gel puriWed and religated to itself by reacting 24 ng vector
DNA with 200 U of T4 DNA ligase for 2 h at room temperature. The religated vector was then electroporated into
Escherichia coli TG1 cells.
Automated Fab ELISA
The prepared Fab expression DNA (pMID21) from
each selection output was transformed into electrocompetent E. coli (cat. no. 200123, Stratagene) and plated onto
DY52 plates (2£ YT agar, 2% glucose, 100 g/ml ampicillin). Colonies were then picked and dispensed into wells of
96-well plates (each containing 200 l 2£ YT, 2% glucose,
100 g/ml ampicillin) using a robotic colony picker (AutoGen). The individual clones (4608 colonies distributed
among 48 96-well plates) were then grown for approximately 16–18 h at 30 °C, and Fab expression was induced
overnight following the addition of 1 mM isopropyl--D-1thiogalactopyranoside (IPTG). A 70-l aliquot of the overnight culture was then transferred using a PlateTrak (or a
MiniTrak) to a 384-well assay plate that contained 5 ng/
100 l biotinylated hK1 captured on streptavidin-coated
wells and blocked with 1% bovine serum albumin (BSA).
Colonies expressing Fabs that bind hK1 were identiWed following the addition of anti-Fab coupled to horseradish
peroxidase (Pierce), the addition of 3,3⬘,5,5⬘-tetramethylbenzidine (TMB) substrate (KPL), and absorbance detection at 630 nm using a Tecan Spectra Xuorescence plate
reader. ELISA plate movements were detected by a deXuorescence plate reader, and between reagent additions the
plates were washed using a Biotek plate washer (model
ELx405 Select). ELISA signal/background ratios were
determined by comparison of the signal of each individual
colony to that obtained for a streptavidin-coated well. A
signal/background ratio of 2.4 was used to identify an even
12 96-well plates (1152 colonies) to analyze by DNA
sequencing. DNA sequences of the Fab hits were determined using an Applied Biosystems 3700 DNA sequencer
according to the manufacturer’s instructions.
244
High-throughput aYnity ranking of antibodies / D. Wassaf et al. / Anal. Biochem. 351 (2006) 241–253
High-throughput Fab expression and puriWcation
Flexchip SPR analyses
Bacterial stocks containing the pMID21 plasmid and
expressing unique Fab genes as determined by DNA
sequencing were Wrst grown in 500 l 2£ YT medium (containing 100 g/ml ampicillin and 2% glucose) overnight at
30 °C or for 4 h at 37 °C in 96-well deep-well plates sealed
with air-permeable covers (900 rpm in a Multitron plate
shaker/incubator). New 24-deep-well plate cultures were
inoculated with a 1:100 dilution of the stock culture in 6 ml
2£ YT medium (containing 100 g ampicillin/ml and 0.1%
glucose) per well. The cells were allowed to grow at 37 °C
while shaking at 380 rpm until the OD600 was 0.8–1.0
(» 2 h). Fab expression was then induced by the addition of
1 mM IPTG and an overnight growth at 30 °C with shaking. Cells were then centrifuged in the 24-well plates at
4000g for 15 min at 4 °C using a plate centrifuge (Eppendorf 5804R).
Supernatants from four 24-well plates were reformatted to the 96-well plates using a liquid handler with liquid
sensing (Tecan Genesis Workstation 200). To process as
much supernatant as possible (»5.8 ml), it was necessary
to distribute the volume among four replicate 96-deepwell plates, where each well contained approximately
1.45 ml supernatant. The four replicate 96-well plates were
then placed on the MiniTrak 5 automation deck (CCS
Packard MiniTrak 5) to process the 96 diVerent Fabs
simultaneously using protein A containing PhyTip columns (PhyNexus). The Fabs were captured on the protein
A in the PhyTip columns by a series of 10 aspiration (5 l/
s) and dispense (5 l/s) cycles and then were washed with
170 l phosphate-buVered saline (PBS) (2 aspiration and
dispense cycles) using the MiniTrak 5. Fabs were eluted
twice with 20 l PhyTip elution buVer (50 mM sodium
phosphate, 150 mM sodium chloride, pH 2.5) and neutralized with 12 l PhyTip neutralization buVer (1 M Hepes).
Fab concentrations were determined by absorbance at
280 nm in a 96-well UV plate (Costar) using the Spectramax Plus plate reader (Molecular Devices), and where
1 mg/ml Fab yielded an absorbance of 1.4, Fab purity was
analyzed by loading 1.5 g Fab onto a 4–20% SDS–PAGE
gel (Invitrogen).
The printed AYnity Chip was inserted into the Flexchip
SPR device, and the steps recommended by the manufacturer (Biacore) were followed. First, regions of interest
(ROIs) on the chip that contain printed Fab and corresponding reference spots were assigned while the chip was
dry. Each ROI has associated reference spots that surround
the ROI and were used to account for bulk refractive index
changes as well as corrections due to instrumental drift. The
chip was then blocked with 2.5% Wsh gelatin (Sigma) for
5 min at a Xow rate of 1 ml/min prior to the start of the run
to prevent nonspeciWc analyte (hK1) adsorption to the chip
surface. The blocked chip was then equilibrated with PBST
(PBS containing 0.01% Tween 20) under Xow (1 ml/min) for
approximately 1 h or until the baseline drift was considered
to be acceptable (<2 resonance change units [RCU]/h). The
analyte (100 nM hK1) was Xowed over the equilibrated
chip surface for 7 min at a rate of 1 ml/min to observe the
association phase of each Fab–hK1 interaction. The analyte injection was followed by a 7-min wash with PBST to
observe the dissociation phase of each Fab–hK1 interaction. The change in SPR signal was monitored at 25 °C. The
method does not require regeneration. Data analysis was
performed by using either the Flexchip data analysis package provided with the instrument or the CLAMP program
[28]. The triplicate sensorgrams obtained for each Fab producing an acceptable signal (>5 RCU) were globally Wt to a
1:1 interaction model with mass transport correction to
provide kon and koV estimates.
Fab printing procedure
Up to 96 diVerent Fabs were printed in triplicate
directly onto the gold surface of an AYnity Chip (Biacore) using a Cartesian spotter (Genomic Solutions). The
printing was done in contact mode using an 8-m stainless-steel pin (cat. no. SMP8B, Genomic Solutions) at
room temperature under 80% relative humidity. PuriWed
Fabs were printed directly from the neutralized PhyTip
elution buVer at the concentrations obtained without further dilution to produce a 16 £ 18 array for use in the
Flexchip SPR device. Each printed chip was dried for 1 h
at room temperature and assembled as described by the
manufacturer.
Biacore 3000 SPR analyses
A Biacore streptavidin chip was used to immobilize biotinylated hK1 that was diluted into HBS–EP (10 mM
Hepes [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.005% P20)
on Xow cell 2 (RL D 30 RU), Xow cell 3 (RL D 56 RU), and
Xow cell 4 (RL D 113 RU). Flow cell 1 did not contain hK1
and was used for reference correction. The Fabs were
diluted 4.0, 7.8, 15.6, 31.25, 62.5, 125, and 250 nM in PBST
buVer (5.5 mM phosphate [pH 7.4], 0.15 M NaCl, 0.01%
Tween 20 [v/v]) and injected at 30 l/min for 4 min using the
Kinject program. Following a 20-min dissociation, any
remaining Fab was stripped from the surface with one 8-s
QuickInject of 50 mM citrate (pH 3.0) at 75 l/min followed
by a 30-s injection of running buVer. Sensorgrams obtained
using the above seven concentrations of Fab were globally
Wt to the 1:1 model of interaction using the BIAevaluation
3.2 software.
IC50 measurements
Steady-state enzyme inhibition assays were performed in
black, 96-well round-bottomed microplates in a total volume of 100 l reaction buVer (20 mM Tris [pH 7.5], 150 mM
NaCl, 1 mM EDTA, 0.1% PEG, 0.1% Triton X-100) containing 5 nM hK1, a varied concentration of Fab, and
High-throughput aYnity ranking of antibodies / D. Wassaf et al. / Anal. Biochem. 351 (2006) 241–253
100 M Pro-Phe-Arg–7-amino-4-methylcoumarin (AMC)
substrate. Before the addition of substrate to initiate the
reaction, the hK1 and Fab were incubated in the well of the
assay plate for 1 h at 30 °C. The substrate was then added,
and the reaction was monitored using a Xuorescence plate
reader (Gemini XS, Molecular Devices) using an excitation
wavelength of 360 nm and an emission wavelength of
460 nm.
Results
Phage display selection and screening strategies
The goal of this phage display selection and screening
project was to obtain high-aYnity antibody inhibitors of
the protease activity of hK1. The selection strategies outlined in Fig. 1 yielded Wve diVerent phage outputs. From the
phage outputs, a total of 4608 recombinant Fab fragments
were screened by ELISA for binding to immobilized hK1.
ELISA analysis identiWed 1152 Fabs as hits based on signal/background ratios greater than 2.4. The ELISA was
performed using Fab fragments rather than phage to identify Fabs that not only bound the target but also were functionally expressed. DNA sequencing of the heavy and light
chains of the 1152 Fab hits identiWed 355 unique Fab
sequences. We developed a rapid method that combines
automated Fab puriWcation and analyses using the Flexchip SPR instrument to screen these Fabs and obtain estimates of the kinetic constants of all the unique anti-hK1
Fabs discovered in this project.
Automated Fab puriWcation
Escherichia coli-conditioned medium containing
expressed Fabs was processed using protein A PhyTip col-
245
umns on a liquid handler that are capable of processing 96
samples in parallel. The concentrations of the puriWed Fabs
were determined in a 96-well plate by absorbance at 280 nm
(Fig. 2A). The yield of recovered Fab varied widely among
the diVerent Fab clones. However, because the Wnal volume
of the eluted Fabs is low, the concentrations of 228 of the
355 unique Fabs that were processed were suYciently high
for Flexchip analysis (Fig. 2A). To date, we have found that
Fab concentrations equal to or greater than approximately
100 g/ml are required to obtain satisfactory SPR signals
(>5 RCU) using the Flexchip instrument. The purity of representative Fabs is shown Fig. 2B, where a Wxed amount
(1.5 g) was analyzed by SDS–PAGE. The purity of the
recovered Fabs was assessed by SDS–PAGE and was
found, for the majority of Fabs, to be greater than 80%.
From the SDS–PAGE analyses, the absorbance method
appears to overestimate the concentrations of certain Fabs
(e.g., lanes 9 and 13 in Fig. 2B). We have since found that
sensitive protein assays, such as the Quant-It assay (Molecular Probes), provide more accurate estimations of the concentrations of Fab produced using our automated
puriWcation method.
Flexchip validation studies
The accuracy of the kinetic constants obtained using the
Flexchip were assessed by comparison with those values
obtained by Biacore 3000 SPR and with equilibrium IC50
values acquired using steady-state enzyme inhibition (Table
1). The two SPR methods diVered in their experimental
conWgurations. On the Flexchip, the Fab was immobilized
on a gold surface and the hK1 antigen was Xowed over the
surface at a single concentration (100 nM). In contrast, the
experiments on the Biacore 3000 were performed using biotinylated hK1 that was captured on a streptavidin-coated
Fig. 1. Overview of antibody phage display selection strategy. To direct the binding of the phage to the active site of the enzyme, aprotinin, a known active
site binder and inhibitor of hK1, was used in two diVerent selection strategies. In strategy 1, aprotinin was used to elute phage that bound immobilized
hK1 in each round of selection. The approach of strategy 2 was to remove nonactive site binders of hK1 by pretreating the library with immobilized hK1
complexed with aprotinin in each round of selection. Phage that did not bind the immobilized hK1– aprotinin complex were then added to immobilized
active hK1.
246
High-throughput aYnity ranking of antibodies / D. Wassaf et al. / Anal. Biochem. 351 (2006) 241–253
Fig. 2. Analyses of Fabs puriWed by the automated puriWcation method. Fabs were expressed and puriWed in batches of up to 96, as described in Materials
and methods. In panel A, the concentration of each Fab in Wve diVerent puriWcation batches was determined by absorbance at 280 nm, where a 1-mg/ml
Fab solution has an absorbance of 1.4. The purity of all the Fabs was evaluated by using nonreducing SDS–PAGE. Panel B shows the analyses of 14 Fabs
from batch 1 (lanes 2–15) that were puriWed using the automated method. Based on the Fab concentration, 1.5 g of each Fab was loaded into each lane.
The concentrations of the Fabs in lanes 9 and 13 apparently were overestimated by the absorbance method. Lane 1 is a molecular weight marker (Invitrogen) composed of myosin (188 kDa), phosphorylase B (98 kDa), BSA (62 kDa), glutamic dehydrogenase (49 kDa), alcohol dehydrogenase (38 kDa), carbonic anhydrase (28 kDa), myoglobin red (17 kDa), lysozyme (14 kDa), aprotinin (6 kDa), and insulin B chain (3 kDa).
Table 1
Comparison of Flexchip, Biacore 3000, and enzyme inhibition data
Fab
Flexchip
kon (M
M0107-D12
M0114-G06
M0098-E09
M0093-F09
M0112-D07
M0113-G11
M0102-C02
Biacore 3000
¡1 ¡1
4.9 £ 105
1.4 £ 105
7.3 £ 105
9.4 £ 105
1.0 £ 105
8.9 £ 105
1.6 £ 105
s )
¡1
¡1 ¡1
koV (s )
Kd (nM)
kon (M
6.3 £ 10¡4
1.9 £ 10¡4
1.7 £ 10¡3
3.4 £ 10¡3
7.7 £ 10¡4
1.9 £ 10¡2
3.8 £ 10¡3
1.3
1.4
2.3
3.6
7.7
22
24
4.2 £ 105
2.4 £ 105
2.1 £ 106
1.8 £ 106
3.5 £ 105
2.5 £ 105
5.0 £ 105
s )
¡1
koV (s )
Kd (nM)
2.5 £ 10¡3
3.1 £ 10¡4
1.6 £ 10¡3
2.2 £ 10¡3
6.8 £ 10¡4
1.3 £ 10¡2
1.1 £ 10¡2
5.8
1.3
0.72
1.2
1.9
24
21
Enzyme inhibition
IC50 (nM)
1.7
1.2
2.5
2.5
N.I.
N.I.
N.I.
Note. N.I., no inhibition observed at the highest concentration of Fab tested (120 nM).
surface and the Fab was Xowed over the surface at diVerent
concentrations. The SPR data acquired using both instruments were Wt to a 1:1 interaction model using the data
analysis software supplied with each instrument (Table 1)
and using CLAMP (data not shown) [28]. Overall, both
SPR methods identiWed the same highest aYnity Fabs
(Kd 6 10 nM) and the same lowest aYnity Fabs
(Kd 7 10 nM). The calculated Kd values acquired using the
High-throughput aYnity ranking of antibodies / D. Wassaf et al. / Anal. Biochem. 351 (2006) 241–253
two SPR methods all were within 5-fold of each other, and
three of the seven Fabs examined (M0114-G06, M0113G11, and M0102-C02) had Kd values within 20% of each
other. With two exceptions (M0113-G11 and M0102-C02),
kon values measured on the Biacore 3000 were between 1.7fold (M0114-G06) and 2.9-fold (M0098-E09) higher than
the kon values measured on the Flexchip. With three exceptions (M0093-F09, M0112-D07, and M0113-G11), the koV
values were up to 4-fold faster (M0107-D12) when measured on the Biacore as compared with the Flexchip. The
calculated Kd values obtained from both SPR methods
were compared with the IC50 values for the six anti-hK1
Fabs that were found to inhibit the protease activity of hK1
(Table 1). The IC50 values were within 4-fold of the calculated Kd values obtained using both SPR methods. From
this set of data, it is apparent that both SPR methods
obtain similar estimates of kinetic constants.
The reproducibility of the kinetic constants obtained
using the Flexchip and the inXuence of spot location on the
chip on the observed kinetic constants were examined by
preparing a gold chip composed of 242 spots of a single
Fab (M0131-F07). The binding sensorgrams were observed
by Xowing a single concentration of hK1 (100 nM) over the
chip surface. Data were analyzed by creating 23 groups,
each of which was composed of two diVerent concentrations of spotted antibody fragment (500 and 250 g/ml) in
triplicate, resulting in six sensorgrams per group, as shown
in Fig. 3A. The six sensorgrams in each data analysis group
were globally Wt to the model for a 1:1 interaction with a
local mass transport term (km) that was allowed to Xoat
between spots [22]. The average kon value among all data
analysis groups was 2.5 £ 105 M¡1 s¡1 with 11% coeYcient
of variation (CV), and the average koV was 7.3 £ 10¡4 s¡1
with 14% CV. The plots of kon and koV values versus their
analysis group indicate that the estimates of kinetic constants obtained using the Flexchip do not appear to be
dependent on spot location on the chip (Figs. 3B and C).
The inXuence of experimental orientation on the
observed kinetics was also investigated by inverting the
analyte and immobilized ligand. When each hK1 antigen
concentration (0.5 and 0.25 mg/ml) was printed in triplicate
on a gold chip and the M0131-F07 Fab (100 nM) was
Xowed over the surface at a rate of 0.5 ml/min, the average
kon was 1.1 £ 105and the average koV was 1.3 £ 10¡3 (data
not shown). These estimates for kon and koV are within 2.4and 1.3-fold, respectively, of the kinetic constants obtained
in the orientation where the Fab is immobilized and the
hK1 antigen is Xowed over the surface (see above).
Flexchip aYnity ranking
PuriWed unique Fabs were printed directly from the neutralized PhyTip elution buVer on gold AYnity Chips in
triplicate and in groups of up to 96 diVerent Fabs per chip.
Estimates of the kinetic association and dissociation constants for each printed Fab were obtained by Xowing hK1
over the chip surface at a single concentration of 100 nM
247
and globally Wtting the three spots for each Fab to the 1:1
interaction model with local Rmax and mass transport
terms. Figs. 4A and B show representative sensorgrams for
two diVerent Fabs that were well Wt by the 1:1 model.
We obtained estimates of kinetic constants for 191 of the
355 unique anti-hK1 Fab fragments that were examined
(Fig. 4C). A wide range of aYnities were observed, with the
highest apparent dissociation constants being greater than
100 nM and the lowest being less than 1 nM (Fig. 4C). The
concentrations of some Fabs (127 of 355) were less than
100 g/ml, which is the level required to produce a satisfactory signal. We deWned a satisfactory signal as equal to or
greater than 5 RCU. The remaining 37 Fabs appeared to be
at suYciently high concentrations for the SPR method but
did not appear to bind hK1 under these conditions.
IdentiWcation of active site binding Fabs
Anti-hK1 Fabs that bound the active site of hK1 were
identiWed by comparing sensorgrams obtained by Xowing
free hK1 to those obtained by Xowing hK1–aprotinin complex (Fig. 5). Aprotinin is a Kunitz domain and a known
active site inhibitor of hK1 and other serine proteases [29].
Because aprotinin is an active site inhibitor of hK1, those
Fabs that do not bind the hK1–aprotinin complex with
hK1 may be active site binders and potential inhibitors of
hK1 activity. Sensorgrams for a Fab (M0097-G11) that
binds only free hK1 (Fig. 5A) were compared with those for
a Fab (M0135-F03) that appears to bind the hK1–aprotinin complex as well as free hK1 (Fig. 5B). A number of
Fabs (43) were analyzed according to the ratio of the signal
(in RCU) observed from binding the hK1–aprotinin complex to that observed from binding free hK1 (Fig. 5C). Of
43 Fabs examined, 15 bound the hK1–aprotinin complex
with an endpoint signal that was less than 20% of the endpoint signal observed with free hK1. All 43 Fabs were
tested for their ability to inhibit the protease activity of
hK1 toward a synthetic peptide substrate, and 27 were
found to be inhibitors (Fig. 5C).
Discussion
For protein therapeutic discovery programs, the ability
to simultaneously determine the binding properties of
many Fabs in a single experiment oVers a substantial reduction in analysis time, consistency in experimental conditions, and a potential reduction in the amount of target
antigen consumed. The method we have described meets
the above criteria and complements a typical automated
phage display selection and screening process. The Wrst step
in the phage display process is the screening or panning
step during which phage binders to a target protein are
selectively removed from the library. The selected phage are
ampliWed and used for additional rounds of selection and
ampliWcation. To maximize the diversity of selected antibodies, we generally limit the number of rounds and compensate by screening more individual phage isolates (e.g.,
248
High-throughput aYnity ranking of antibodies / D. Wassaf et al. / Anal. Biochem. 351 (2006) 241–253
Fig. 3. EVect of spot position on kinetic constants obtained using Flexchip SPR. The same Fab was printed 242 times at concentrations of 250 and 500 g/
ml on a gold chip. Panel A is an SPR image of the printed chip generated using the Flexchip control software that shows the locations of the data analysis
groups with respect to the Xow inlet and outlet. Analysis groups were created by combining three adjacent spots printed using 250 g/ml Fab with three
adjacent spots printed using 500 g/ml Fab. The six sensorgrams obtained in each data analysis group using a single concentration of hK1 analyte
(100 nM) were globally Wt to a 1:1 interaction model using the Flexchip data analysis software to obtain estimates of the kinetic constants. Panel B shows
the association rate constants (kon) obtained for the data analysis groups. Panel C shows the dissociation rate constants (koV) obtained for the data analysis
groups.
High-throughput aYnity ranking of antibodies / D. Wassaf et al. / Anal. Biochem. 351 (2006) 241–253
249
Fig. 4. AYnity ranking of anti-hK1 Fabs by Flexchip. Up to 96 diVerent anti-hK1 Fabs were spotted on a gold chip surface in triplicate, and sensorgrams
observed with a single concentration of hK1 analyte (100 nM) were globally Wt to a 1:1 interaction model using the Flexchip data analysis software to
obtain estimates of the kinetic constants. Panel A shows triplicate sensorgrams (thin lines) obtained from three diVerent spots of a high-aYnity anti-hK1
Fab (M0114-G06) that were globally Wt (thick line) to yield estimates of the kinetic constants describing the interaction (kon D 1.4 £ 105 M¡1 s¡1,
koV D 1.9 £ 10¡4 s¡1). Panel B shows triplicate sensorgrams (thin lines) of a moderate-aYnity anti-hK1 Fab and the Wt (thick line, kon D 4.1 £ 105 M¡1
s¡1,koV D 1.9 £ 10¡2 s¡1). Panel C displays the kon and koV values for the 191 anti-hK1 Fabs for which estimates of the kinetic constants were obtained.
thousands of isolates) using an automated method such as
an ELISA with immobilized antigen. Isolates with a high
ELISA signal are submitted for DNA sequencing to identify unique antibodies (e.g., hundreds). Once the unique
antibodies are batch cloned as Fabs in an expression vector
and recovered by additional DNA sequencing, they are
ready to be analyzed by the method described here. The
method involves Fab expression in 24-well plates, puriWcation by protein A PhyTip columns, and aYnity determination by Flexchip SPR.
The highly parallel capabilities of the Flexchip instrument are best exploited when the target antigen is Xowed
over the immobilized Fabs. The kinetic constants obtained
in this experimental conWguration are based on the concen-
tration of a single antigen solution and thereby obviate the
need for a multitude of accurate concentrations of active
antibody fragment [30,31]. A limitation of this restricted
experimental conWguration occurs when multimeric antigens are Xowed over the Fabs, which may lead to avidityinXuenced kinetics that do not Wt well to 1:1 interaction
models. For multimeric antigens, the method described
here may still provide an eVective aYnity ranking method
for antibody phage display, but the estimates of the kinetic
constants may be less accurate. A similar limitation was
observed when antibodies were captured on a Biacore chip
and antigen Xowed over the surface [21]. Fab aYnities
toward multimeric antigens may best be screened using
immobilized or captured antigen with Fab in solution [32],
250
High-throughput aYnity ranking of antibodies / D. Wassaf et al. / Anal. Biochem. 351 (2006) 241–253
Fig. 5. IdentiWcation of active site binders by Flexchip. The ability of multiple Fabs to bind either hK1 or the hK1–aprotinin complex was determined by
comparing sensorgrams observed on introducing either 100 nM free hK1 (solid line) or an hK1–aprotinin complex (dotted line) that was prepared by mixing 100 nM hK1 with 1 M aprotinin. Panel A shows the observed sensorgrams for the M0097-G11 Fab, which appears to be an active site binder. Panel
B shows the observed sensorgrams for the M0135-F03 Fab, which appears to be a nonactive site binder. The signals at the end of the injections of either
free hK1 or hK1–aprotinin complex were recorded, and the bound/free signal ratios for all 43 Fabs tested are shown in panel C. Fabs found to inhibit hK1
protease activity are represented by gray bars, and Fabs that did not appear to inhibit the activity toward the synthetic substrate are represented by black
bars.
although this approach requires knowledge of the active
concentrations of each Fab and becomes a more time-consuming sequential method.
The diVerences in the Flexchip signal intensities among
the diVerent Fabs are not unexpected and may be attributed to factors such as (i) diVerences in Fab aYnity for
hK1, (ii) the varied concentrations of printed Fabs, (iii)
overestimated Fab concentrations, (iv) variability in the
amounts of Fab printed by the contact spotter, and (v)
diVerences in the amounts of correctly oriented Fab on the
chip surface. We have observed that to obtain a satisfactory
Flexchip SPR signal (>5 RCU), a Fab concentration equal
to or greater than 100 g/ml usually is required. Because
approximately 10 l is required to submerge the pin, the
Flexchip method described here requires approximately
1 g Fab, although it consumes only a small fraction to
print the chip. In comparison, a similar experiment performed using immobilized Fab on a Biacore 3000 platform
would require approximately 100 l of a 5-g/ml protein
solution (0.5 g).
Fabs printed on gold may result in a surface of randomly oriented Fab molecules, and some of these orientations may interfere with antigen binding. However, our
studies to date comparing oriented Fab immobilization
High-throughput aYnity ranking of antibodies / D. Wassaf et al. / Anal. Biochem. 351 (2006) 241–253
using speciWc capture surfaces (anti-cMyc or protein A)
have not yet identiWed Fabs that bind only when speciWcally captured (data not shown). The immobilization of
recombinant Fabs on gold surfaces may be predominantly
mediated by an interaction between an accessible protein
disulWde and gold [33]. In fact, the recombinant Fabs used
here have C-terminal cysteine residues on both the heavy
and light chains that form an interchain disulWde. This
disulWde, unlike the disulWde with the immunoglobulin G
domains, is expected to be accessible for interaction with
gold and may produce a speciWcally oriented Fab surface,
consistent with the observation that the majority of our
sensorgrams Wt well to a 1:1 model of interaction (Fig. 3).
Further studies are required to investigate the mode of
interaction between recombinant Fab and a gold surface.
The Flexchip method will beneWt from advances in surface
chemistries and speciWc capture surfaces.
The kinetic constants obtained for seven Fabs using the
Flexchip were compared with those obtained using Biacore
3000 SPR and to equilibrium IC50 values measured by
steady-state enzyme inhibition (Table 1). The kon values
obtained for Wve of the seven Fabs using the Biacore analyses were signiWcantly faster than those obtained by Flexchip analyses. This diVerence may suggest that some of the
measurements obtained using Flexchip are either mass
transport limited or subject to steric eVects arising from the
Fab immobilization on gold. However, mass transport
eVects are expected to inXuence estimates for both the kon
and koV values [34–36]. The observation that the koV values
between the two diVerent SPR methods agree well (Table 1)
and the observation that similar kinetic constants were
obtained using two diVerent Xow rates (0.5 and 1 ml/min
251
[data not shown]) suggest that the diVerences in the kon values between the two SPR instruments are not attributable
to mass transport eVects.
Phage display has been shown previously to be an eVective method to obtain antibodies against speciWc epitopes
of an antigen [37]. As shown in Fig. 1, aprotinin was used in
the phage display selection either as an elution agent (outputs 1–4) or for depletion with the hK1–aprotinin complex
(output 5). The goal of both approaches was to obtain antibodies that bind at the active site of hK1 and are inhibitors
of the protease activity of hK1. By comparing the signal
intensity observed with free hK1 with that observed with
the hK1–aprotinin complex, we were able to identify antibodies that appear to either bind at the active site or no
longer bind the complex (Fig. 5). The observation that
many of the inhibitors bind similarly to the hK1–aprotinin
complex and free hK1 suggests that these inhibitors are
binding outside of the active site, possibly via a noncompetitive inhibition mechanism. However, further studies are
required to determine the steady-state enzyme inhibition
mechanisms of these inhibitors. The Wnding that several
noninhibitory Fabs did not bind the hK1–aprotinin complex may indicate that these Fabs are binding near the
active site but do not prevent enzymatic hydrolysis of the
small peptide substrate. This type of competition experiment could also be used in other applications to rapidly
identify antibodies that bind a particularly important epitope provided that reagents are available to block the site
of interest on the antigen.
In addition to the above-described Flexchip competition
experiments using the hK1–aprotinin complex, we screened
several Fabs for hK1 inhibition using a single Fab concen-
Fig. 6. Evaluation of phage display selection strategies. The calculated equilibrium dissociation constants, as determined by Flexchip analyses, for each
Fab is plotted against the selection output (see Fig. 1), as shown by the open circles. Those Fabs that bound hK1 and inhibited its protease activity were
characterized by steady-state enzyme kinetics to obtain IC50 estimates (crosses).
252
High-throughput aYnity ranking of antibodies / D. Wassaf et al. / Anal. Biochem. 351 (2006) 241–253
tration (120 nM) and a synthetic peptide substrate. Those
Fabs that inhibited hK1 were then further characterized by
the determination of IC50 values. The Fabs were grouped
according to the selection output from which they originated (Fig. 1) and were plotted against their Kd and IC50
values (Fig. 6). Knowledge of kinetic constants for such a
large number of antibodies guides the phage display screen
toward the discovery of the highest aYnity antibodies and
can provide useful information about which selection strategy is most eVective. Although high-aYnity hK1 antibodies
were obtained from all selection outputs, it is evident that
inhibitors were obtained only in those outputs that used
aprotinin either to elute bound phage or to deplete the
library of phage that bound outside of the active site. For
example, output 3 consisted of phage that remained associated with immobilized hK1 after two rounds of elution
with aprotinin and did not contain any hK1 inhibitors.
In summary, the method described here combines two
novel technologies, high-throughput antibody puriWcation (PhyTip) and microarray-based SPR (Flexchip), to
constitute an eVective secondary screen for antibody
phage display. The method provides accurate estimates of
the aYnities of all the unique antibodies discovered by an
automated phage display program. We demonstrated a
powerful application of the method in its ability to rapidly group up to 96 diVerent antibodies according to epitope
(aprotinin
competition
experiment).
We
demonstrated the utility of the method by ranking the
aYnity of nearly 200 diVerent antibodies directed toward
hK1, leading to the identiWcation of antibodies with subnanomolar aYnities. In combination with a human Fab
phage display library engineered to yield a diverse repertoire of binders, the method can be used to sort rapidly
through hundreds of hits to identify the most potent leads
for drug development.
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