Analytical Biochemistry 609 (2020) 113974

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Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio

Binding affinity determination of therapeutic antibodies to membrane

protein targets: Kinetic Exclusion Assay using cellular membranes for
anti-CD20 antibody
Amit Vaish a, Joanne S. Lin b, Helen J. McBride c, Peter J. Grandsard a, Qing Chen a, *
a
Discovery Attribute Sciences, Discovery Research, Amgen Inc., Thousand Oaks, CA, USA
b
Eli Lilly and Company, Lilly Biotechnology Center, San Diego, CA, USA
c
Technology Transfer and Corporate Partnerships, California Institute of Technology, CA, USA

A B S T R A C T

Antibody-based therapeutics targeting membrane proteins have evolved as a major modality for the treatment of cancer, inflammation and autoimmune diseases.
There are numerous challenges, ranging from desired epitope expression to reliable binding/functional assays which are associated with developing antibodies for
this target class. Specifically, having a robust methodology for characterizing antibody interaction with a membrane protein target is essential for providing guidance
on dosing, potency and thus expected efficacy. Fluorescence-activated cell sorting (FACS) has been commonly used to characterize antibodies binding to membrane
protein targets. FACS provides information about the antibody-receptor complex (antibody bound to cells) and the apparent equilibrium dissociation constant (KD ) is
′

elucidated by fitting the antibody-receptor binding isotherm as a function of total antibody concentration to a nonlinear regression model. Conversely, Kinetic
Exclusion Assay (KinExA) has been used to measure solution-based equilibrium dissociation constant (KD) of antibodies. Here, KD is determined by measuring the free
antibody concentration at equilibrium in a series of solutions in which the antibody is at constant concentration and the receptor (either in the membrane or the cell)
is titrated. We measured the binding affinity of the anti-CD20 antibody, Rituximab, using both FACS and KinExA. There was ~25-fold difference in the binding
affinity measured by these two techniques. We have explored this discrepancy through additional experiments around the mathematical framework involved in the
analysis of these two different binding assays. Finally, our study concluded that KinExA enables accurate measurement of the KD for strong protein-protein in­
teractions (sub-nanomolar values) compared to FACS.

1. Introduction membrane protein binding characterization for antibodies and present a

Recombinant antibodies have emerged as an important therapeutic
modality for the treatment of various diseases through neutralization or
activation of target molecules ranging from growth factor receptors to
soluble cytokines [1–5]. Many next generation antibody therapeutics
are targeted to membrane proteins [6]. This class of targets has posed a
perpetual challenge from preparing membrane protein antigens for
antibody generation to binding characterization. Developing a robust
method for evaluating binding affinity of therapeutic antibodies against
membrane protein targets is important for drug discovery as it provides
the guidance on dosing and expected efficacy. The affinity also has
implications for other desired properties of an antibody including target
method for the accurate determination of affinity.
Fluorescence-activated cell sorting (FACS) has been commonly used
to characterize antibodies binding to membrane protein targets [7,8]. In
this technique, antibody is titrated against a fixed concentration of cells
expressing the target, followed by incubation, rinsing, and finally
exposing all the samples to a fluorescently-labeled secondary antibody
for measuring fluorescence intensity, which corresponds to the amount
of primary antibody that remains bound on the cell surface. FACS pro­
vides information about the antibody-receptor complex (antibody
bound to cells), but not pertaining to the free antibody and antigen
concentrations in the binding assay. The apparent equilibrium dissoci­
ation constant (KD ) can be elucidated by fitting the antibody-receptor
′

internalization and recycling, effector function across different levels of binding isotherm as a function of total antibody concentration to a
cell expression, and inhibition of signaling. Here, we present a gener­ nonlinear regression model.
alized methodology to overcome some of the traditional limitations of This model makes two assumptions, first that the cells have reached

Abbreviations: CBP, Constant Binding Partner; CBPC, Constant Binding Partner Concentration; FACS, Fluorescence-Activated Cell Scan; KinExA, Kinetic Exclusive
Assay; KD, Equilibrium Dissociation Constant; K’D, Apparent Equilibrium Dissociation Constant.
* Corresponding author.
E-mail addresses: qchen@amgen.com, qchen@amgen.com (Q. Chen).

https://doi.org/10.1016/j.ab.2020.113974
Received 22 January 2020; Received in revised form 15 September 2020; Accepted 18 September 2020
Available online 30 September 2020
0003-2697/© 2020 Elsevier Inc. All rights reserved.
A. Vaish et al.
equilibrium with the titrated primary antibody, and second that the
concentration of cell bound antibody is an insignificant fraction of the
total antibody concentration [9]. These assumptions can only be true
when the total concentration of antigen expressed on the cell surface is
less than the equilibrium dissociation constant, KD. However, due to the
sensitivity limitations, accurate FACS measurements are difficult to
obtain for strong interactions involving sub-nanomolar KD values.
The Kinetic Exclusion Assay (KinExA), developed by Sapidyne, has
evolved as a leading biophysical technique to measure solution-based
equilibrium dissociation constant (KD) of high-affinity binding part­
ners [10–12]. In KinExA, KD is determined by measuring the free anti­
body binding sites at equilibrium in a series of solutions in which the
antibody at a constant concentration is titrated with the binding partner.
For soluble proteins, KinExA is a relatively straightforward tool to study
binding interactions, since the concentration of a soluble protein target
can be accurately controlled. Here, the equilibrated solutions containing
doubly unliganded antibody and singly liganded antibody are intro­
duced to a ligand-immobilized solid phase (beads) for binding charac­
terization [13,14]. Considering the bivalency of monoclonal antibodies,
the total antibody concentration used in KinExA binding model is the
sum of doubly unliganded antibody with 2 free binding sites, singly
liganded antibody having 1 free binding site, and doubly liganded
antibody [15]. In case of membrane targets, not only is the concentra­
tion of membrane proteins often unknown, it is also required to remove
the membrane containing fractions from the equilibrated solutions
before measuring concentration of the membrane free antibody. This
separation process leads to removal of singly liganded antibody from
doubly unliganded antibody in testing samples. To account for the dif­
ference between the sampling in analysis of antibodies for soluble pro­
tein and membrane protein targets, Sapidyne provided “whole cell
equilibrium binding” model in the KinExA-Pro software. For years,
KinExA has provided a reliable methodology [16] to determine KD of
antibodies for binding with membrane targets in their natural environ­
ments using whole cells [3,4,17,18]. However, it is imperative to have a
consistent receptor expression level in the cells used to obtain a repro­
ducible binding affinity determination. We, along with others, have
observed that the number of times cells have been transferred
batch-to-batch during cell culture, which is commonly referred as
“passage number”, can decrease the membrane protein expression level
significantly [19]. To circumvent this issue and ensure reproducibility,
we isolated and froze a large quantity of target-bearing membranes at
− 80 ◦ C for use in antibody titration experiments using KinExA.
Here we describe an integrated methodology to determine KD using
isolated cell membranes containing target proteins for analysis on the
KinExA platform (Fig. 1). Traditional FACS analysis was used to measure
target protein density on the cell-surface prior to membrane prepara­
tion, and to measure KD . By comparing KD and KD for the same pair of
′ ′
binding partners, we describe conditions under which an accurate KD
can be determined and demonstrate that KinExA is the most appropriate
method for accurate KD determination of tight binding molecules.
Fig. 1. Schematic illustration of KinExA-based binding affinity determination using cellular membrane. Cells were quantified via FACS for receptor expression,
followed by membrane isolation, and KinExA-based binding analysis.
2
Analytical Biochemistry 609 (2020) 113974
2. Results and discussions
2.1. Quantification of target protein expression on the cell surface using
FACS
Membrane-associated protein CD20 was used as the target for
binding analysis with the anti-CD20 antibody, rituximab [20]. Ritux­
imab is a chimeric mouse/human therapeutic monoclonal antibody
approved for the treatment of B-cell derived lymphoma [20]. It binds to
CD20, a ~35 kD transmembrane protein found on the surface of a subset
of B-cells. FACS analysis was performed to measure the number of CD20
molecules per WIL-2 cell as a function of passage number using
fluorescently-labeled rituximab. Fig. 2A displays FACS spectra of CD20
expression on WIL-2 cells after the first harvest (passage number 0). 10
or 20 μg/mL of Alexa 488-labeled rituximab was used to determine
maximum fluorescence shift from that of untreated cells. The similar
intensity shift of WIL-2 cells incubated with 10 or 20 μg/mL Alexa
488-labeled rituximab indicates that the CD20 on cell surface was
saturated by the labeled antibody. As the negative control, incubation of
these cells with an Alexa 488-labeled non-target antibody resulted in a
minimal intensity shift, demonstrating the specificity of Alexa
488-labeled rituximab binding to the target antigen. As shown in Fig. 2B,
the mean intensity of anti-CD20 bound cells (geo-mean) was used in the
calibration curve derived from standards to calculate antibody binding
capacity (ABC), which is essentially the number of receptors per cell.
ABC decreases with passage number as illustrated in Fig. 2C. While cell
viability at harvest remained at 91%, cell-surface CD20 density dropped
to ~50% by the 6th passage. Reported previously [18], this kind of
variability in expression level can directly impact the reproducibility
and reliability of the KD determination. To minimize the variability of
antigen concentration presented in the cell binding analysis, we pre­
pared isolated cellular membranes from a large batch of
FACS-characterized cells for long term storage and repeated binding
analyses that are required for regulatory registration of therapeutic
molecules. Using the same batch of the CD20 containing cellular
membranes, we established a streamlined workflow of binding affinity
determination of membrane protein targets using KinExA.
2.2. Measurement of actual equilibrium dissociation constant (KD) of
Rituximab using a Kinetic Exclusion Assay
Following a modified version of the typical KinExA whole cell
analysis method [17,18], we titrated a fixed concentration of antibody
into tubes containing a serial dilution of CD20 membrane. After over­
night incubation (>16 h) at room temperature for the reactions to
approach equilibrium, the incubated samples are subjected to centrifu­
gation and filtration process to separate free antibody from membrane
and membrane-bound antibodies. This KinExA-based process allows us
to determine the membrane free antibody concentration in a binding
reaction that is equilibrated with varying concentrations of
antigen-bearing cell membranes. This process was repeated with four
different antibody concentrations, and the concentrations of the free
antibody from the equilibrated solution were fitted globally for KD
A. Vaish et al.
Fig. 2. Analysis of cell-surface receptor density using the Quantum Simply Cellular (QSC) kit. (A) Alexa Fluor 488-labeled anti-CD20 monoclonal antibody (Rit­
uximab) was used to generate histograms for both CD20 cells and quantified anti-huFc coated reference beads under identical incubation conditions. (B) A calibration
curve was obtained using the reference beads binding to 488-labeled Rituximab for antibody binding capacity (ABC) quantification. (C) Cell-surface CD20 density as
a function of passage number was obtained by calculating ABC from the calibration curve for different passage numbers.
determination using the “whole cell equilibrium binding” model pro­
vided in the KinExA-Pro software. The model takes into account the
bivalency of monoclonal antibodies, which can exhibit mono and
bivalent binding with their targets [18].
As shown in Fig. 3, serially diluted CD20 membrane was titrated
against four different concentrations of rituximab (10, 30, 80, and 200
pM) to generate four independent sigmoidal curves covering a wide
potential affinity range. All these curves were generated using the same
set of serially diluted CD20 membrane concentrations, which enabled
global fitting using n-curve analysis with the “whole cell equilibrium
binding” model for reliable KD determination. In contrast to the model
for soluble protein targets, where global fitting is performed using ti­
trants concentration, “whole cell equilibrium binding” model globally
fits data with the four total antibody concentrations and the fixed serial
membrane dilution ratio of the four titration curves to derive KD and the
receptor expression level. KD of rituximab was determined as 79 pM with
a 95% confidence interval of 50–115 pM. The CD20 expression level was
estimated as 1.5e5 receptors/cell, with a 95% confidence interval of
1.0e5 – 2.2e5 receptors/cell.
2.3. Determination of apparent equilibrium dissociation constant (KD ) of
′
Rituximab using a Flow Cytometry Assay
The apparent equilibrium dissociation constant of rituximab has
been reported at ~5 nM either using radioligand [21] or
fluorescence-based [22] cell binding assays. The KD of rituximab
determined at 79 pM is over 50-fold lower compared to the previously
reported value. To resolve this discrepancy, we performed FACS analysis
mimicking methodology used in the previous reports.
WIL2-S lymphoma cells were cultured and harvested following the
recommended ATCC protocol (ATCC CRL-8885™). The harvested cells
were first measured for CD20 expression as 2.9e6 CD20/cell using the
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Analytical Biochemistry 609 (2020) 113974
method described above (Fig. 2B). FACS analysis was performed using
106 cells/ml. The CD20 receptor concentration was calculated as 4.8 nM
using the following equation.
(Cell number/ml) X (Receptors/Cell)
Receptor Concentration = (1)
Avogadro’ s constant
Fig. 4 shows rituximab binding after four hours incubation at room
temperature with the cells containing 4.8 nM CD20. Data fitting using a
one-site binding model (Y=Bmax*X/(KD +X)) or a dose-response model
′
(Y=Bottom+(Top-Bottom)/(1 + 10^((LogEC50-X)))) available in
GraphPad Prism 7, we obtained 1.9 nM as the value for the apparent
equilibrium dissociation constant (KD ) or the effective antibody con­
′
centration at 50% of antibody/cell complex formation (Fig. 4 B&C).
Here, X is the initial total antibody concentration used in the titration,
and EC50 is the effective antibody concentration eliciting the 50%
binding signal of the maximal response of an antibody-cell complex, and
the calculated KD is the total antibody concentration where half the
′
receptor binding sites are occupied by antibodies via forming the
antibody-cell complex.
Comparing receptor expression determined by FACS with the one
derived from the KinExA analysis, we noted that the expression level
calculated by KinExA (1.5e5 receptors/cell) is approximately 5-fold
lower than that measured by FACS analysis (7.8e5 molecules/cell).
We reasoned that the loss of active CD20 might have occurred during the
membrane preparation process, which involved multiple rounds of cell
resuspension and centrifugation to isolate the cell membrane from other
cellular components.
2.4. Why is the KD higher than the actual KD for tight binders?
′
FACS has been widely applied for high-throughput relative binding
affinity comparison of large panels of antibodies [22]. As discussed
A. Vaish et al. Analytical Biochemistry 609 (2020) 113974

Fig. 3. (A) Schematic of KinExA experimental setup: Sample preparation involving incubation of membrane with antibodies for >16 hours at room temperature,
followed by separation of free antibodies from membrane-bound antibodies using centrifugation and filtration. Samples were introduced to a flow cell containing
anti-huFc coated beads. The captured free antibodies were detected by a fluorescently tagged anti-human antibody [IgG (H + L)]. (B) The binding signal is pro­
portional to the concentration of free antibody in solution for different membrane concentration. Rituximab at four different concentration (10 pM, 30 pM, 80 pM,
and 200 pM) was titrated against CD20 membranes. The top concentration of the membrane contained 2.5 mg/mL total membrane protein. The equilibrium
dissociation constant (KD) was obtained from n-curve “Equilibrium, whole cell” modeling provided in the KinExA™ Pro software.

Fig. 4. (A) Fluorescence intensity of equilibrated

samples containing antibody bound to CD20 cells
was measured by FACS. Binding isotherms of
rituximab titrated onto CD20 cells (antibody
binding capacity (ABC) ~2.9e6/cell) after incu­
bation for four hours. (B) Experimental data in
the form of a rectangular hyperbola curve was fit
with a one-site binding model (Y=Bmax*X/
(KD +X)) to elucidate the apparent equilibrium
′

dissociation constant (KD ). (C) Semi-log graph of

′

experimental date in the sigmoidal shape was fit

with a dose-response model (Y=Bottom+(Top-
Bottom)/(1 + 10^((LogEC50-X))) in GraphPad
Prism 7 to calculate the effective antibody con­
centration at 50% of antibody/cell complex for­
mation (EC50).

4
A. Vaish et al. Analytical Biochemistry 609 (2020) 113974

earlier, accurate FACS analysis requires meeting two assay conditions: ′

(1) equilibrium and (2) a very low ratio of cell bound antibody to total KD
=1+
[Ag]0
(10)
antibody [9]. We tested the impact of less equilibrated samples on KD 2KD
determining KD values using a shorter incubation time at room tem­ Fig. 5 is a simulated plot of KD /KD vs. [Ag]0/KD using eq (10). As
′ ′

perature or slower equilibrium process at 4 ◦ C. Decreasing the illustrated in Fig. 5, KD and KD are similar for the cases where [Ag]0 is
′

cell-antibody incubation duration from four hours to one hour (a typical
incubation time for FACS analysis) at room temperature resulted in
~two-fold increase in the KD from 2 nM as shown in Fig. 4 to about 5 nM
′
less than KD. However, as alluded earlier, FACS sensitivity limitations
requires a critical cell number (~105, which is equivalent to ~5 nM of
CD 20) [9] to generate a measurable signal. As a result, KD supersedes KD
′

(data not shown). Similarly, binding analysis of the Rituximab incubated
at 4 ◦ C for four hours resulted in ~two-fold increase in the KD of 4 nM
′
as [Ag]0 is much higher than KD. Considering KD of rituximab as 79 pM,
the ratio of KD /KD is ~25, which is approximately half of the 50-fold
′

(data not shown). We cannot absolutely confirm that our assay per­ difference with the previously published KD [22]. While no informa­
′

formed at room temperature has reached equilibrium at four hours, but tion was provided on the equilibrium time or cell expression level per­
due to the cell viability constraint this was the longest incubation time
taining to the reported KD , it might be possible that the measurement
′

possible. These data clearly underscore the significance of experimental

was done before equilibrium was achieved which resulted in a higher
conditions in measuring binding affinity.
value of KD than ours.
′

FACS analysis assumes that the KD is equal to the total antibody

′

concentration at which the cell binding is 50% of its maximum value;

however, this is only possible when the bound antibody concentration is
an insignificant fraction of the total antibody concentration. To under­
stand this further, one can consider the concentration of antibody that
results in 50% saturation of cell binding sites as a function of the total
antigen concentration on the cells. The equilibrium dissociation con­
stant (KD) of antibody (Ab) and receptor (Ag) interaction is defined as:
Ab + Ag⇌Ab*Ag
[Ab][Ag]
KD =
[Ab*Ag]
The total concentrations of antibody [Ab]0 and receptor [Ag]0 can be
written for their respective concentration at equilibrium along with the
complex as follows:
[Ab]0 = [Ab] + [Ab * Ag]
[Ag]0 = [Ag] + [Ab * Ag]
The apparent equilibrium dissociation constant (KD ) has been
′
2.5. Constant binding partner concentration plays a key Role in
determining the accurate KD
To further understand the interplay between constant binding part­
ner concentration (CBPC) and KD, we sought to analyze the mathemat­
ical framework involved in KinExA. As described in the previous work
[10,11], rearranging eq (2) to solve for [Ab] and replacing the equilib­
rium concentrations with total concentrations by eqs (3) and (4),
resulted into a quadratic equation as follows:
) √(̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
(2) ( )2
[Ab]0 − [Ag]0 − KD + [Ab]0 − [Ag]0 − KD + 4[Ab]0 KD
[Ab] =
2
(11)
The constant binding partner (CBP) for KinExA is antibody [Ab], and
the free fraction of antibody {[Ab]%} as a function of receptor con­
(3)
centration {[Ag]0} in KinExA can be expressed by eq (11) as:
(4) [Ab]
Free Ab (%) =
[Ab]0

( ) √̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
( )2
defined in the literature [22] for a traditional cell-based assay as the [Ab]0 − [Ag]0 − KD + [Ab]0 − [Ag]0 − KD + 4[Ab]0 KD
concentration of antibody which results in binding of 50% of the re­ =
2[Ab]0
ceptors during titration of antibody against a fixed concentration of
(12)
receptor-containing cells. Adapted from the previous work [11], when
50% of the receptor has been used to form a complex with the antibody; Eq (12) can be further rearranged as:
it results in [Ag]/[Ab*Ag] = 1, therefore eq. (2) can be written as:
KD = [Ab] (5)
At equilibria, substituting eq (5) in eq (3):
KD = [Ab]0 − [Ab * Ag] (6)

At equilibrium, 50% of the receptor have been used to form the

complex [Ab*Ag], therefore eq (4) can be written as:
[Ag]0 = [Ab * Ag] + [Ab * Ag] = 2[Ab * Ag] (7)

After substitution of eq (7) in eq (6) followed by rearrangement:

[Ag]0
[Ab]0 = KD + (8)
2
In eq. (8), [Ab]0 is the total concentration of antibody which resulted
in occupying 50% of the receptor binding sites for eliciting the half
response of the maximal binding signal of antibody-cell complex,
therefore as discussed earlier, this [Ab]0 is the KD
′

[Ag]0
(9)
′
KD = KD +
2
Fig. 5. Simulated curve illustrating the correlation between apparent equilib­
Eq. (9) transforms into a normalized form after dividing it by KD
rium dissociation constant (KD ), actual equilibrium dissociation constant (KD)
′

and receptor concentration [Ag]0. KD surpasses KD for the assay conditions

′

comprising [Ag]0 higher than KD.

5
A. Vaish et al. Analytical Biochemistry 609 (2020) 113974

̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
√( ̅
( ) )2 yeast or phage), where it could measure KD with sub-nanomolar values
′

[Ag]0
1− [Ab]0
− KD
[Ab]0
+ 1 − [Ag][Ab]
0
− KD
[Ab]
+ 4 KD
[Ab] [23,24] using lower number of cells with high surface display levels.
0 0 0
Free Ab (%) = (13) However, binding assay for antibody against a membrane-bound target
2
requires higher number of cells (~105) to generate a measurable signal
Eq (13) can be expanded via Binomial series expansion as discussed in due to the lower expression of these proteins, thus making FACS chal­
Appendix for an approximate solution, which can be used to illustrate lenging to estimate sub-nanomolar KD of antibody/membrane protein
′

Free Ab (%) under two different conditions: interactions.

First, when [Ab]0 ≪ KD, eq (13) can be written as: Conversely, KinExA uses a fixed antibody concentration for titrating
KD against the increasing concentration of target expressing membranes.
Free Ab (%) ≈ (14) Because KinExA is measuring the free antibody binding sites using a
[Ag]0 + KD
fluorescently-labeled secondary antibody, it can detect antibodies at
This expression is a rectangular hyperbola function with KD as a key much lower concentrations (single digit picomolar), enabling genera­
parameter, which makes it a KD-controlled titration curve (Fig. 6). tion of KD-controlled curves by using a lower antibody concentration
Second, when [Ab]0 ≫ KD, and [Ab]0 > [Ag]0 ,Free Ab (%) is expressed than the actual binding affinity. The other advantage of KinExA is its
as: reliance on exact binding equations whereas FACS analysis relies on
measurement of the KD or EC50 which we have shown is only reliable for
′

[Ab]0 − [Ag]0 KD
Free Ab (%) ≈ + (15) KD determination if the constant binding partner concentration is below
[Ab]0 [Ab]0 − [Ag]0
the KD.
And when [Ab]0 < [Ag]0 ,Free Ab (%) is expressed as Recent structural studies have shown that rituximab binds to CD20
dimer on cell surface and forms clustered antibody/antigen complex
KD
Free Ab (%) ≈ (16) [25,26]. Considering the bivalent nature of monoclonal antibody (mAb),
[Ab]0 − [Ag]0
avidity effect could play an important role during binding analysis.
As eqs (15) and (16) are mainly dominated by [Ab]0 , the CBPC, which Using monovalent rituximab Fab one may characterize the avidity of
makes it a CBPC-controlled titration curve (Fig. 6). mAb with comparing to the binding affinity of Fab [17].
In KinExA, binding assay can be modulated in both regimes; KD- In summary, the described methodology from membrane prepara­
controlled and CBPC-controlled titration curves. Global fitting of multiple tion to affinity measurement can be applied to any therapeutic antibody
titration curves that include full or partial KD-controlled ([Ab]0 < 5 KD) against a membrane target. In particular, KinExA enables accurate high-
and CBPC-controlled titration curves derives accurate KD and CBPC affinity antibody KD determination. The accurate affinity determination
values. has broader implications both in the assessment of in vivo target
coverage and for lead selection or engineering efforts in therapeutic
3. Conclusions discovery and development.

Here we have demonstrated use of cellular membranes in combina­ 4. Materials and methods
tion with KinExA for determining the binding affinity of a therapeutic
antibody against a membrane-bound target. Additionally, we measured 4.1. FACS analysis to measure target protein density on the cell surface
prior to membrane preparation
the anti-CD20 antibody, rituximab, KD using FACS, which was compa­
′

rable to the value previously reported. We observed ~25-fold difference

Quantum Simply Cellular (QSC) kit (Cat# 816A) was obtained from
in the binding affinity measured by the FACS and KinExA methods. We
Bangs Laboratories, Inc. The kit consists of five bead populations of one
also provided possible explanations for the difference.
as blank and the other four with coated anti-human Fc antibody at
FACS has been proven to be a viable technique for antibody engi­
specified levels. To measure target specific expression on cell surface
neering and discovery involving cell-surface display platforms (i.e.,
using this kit, it is required to label a fluorophore to an anti-target

Fig. 6. Simulated KinExA graphs depicting

free Ab (%) versus receptor concentration
[Ag] for two [Ab]0, 10 and 1000 pM for an
equilibrium binding affinity (KD) of 100 pM.
The lines represent the simulated KinExA
curves generated by using the exact solution
(eq (12)) describing binding at equilibrium;
solid symbols illustrate the curves generated
using the approximate solution of binding
equation (eq (A6) in Appendix). Blue curve
(left side) illustrates a constant binding
partner [Ab] concentration (CBP) below the
KD, and the shape of the titration curve fol­
lows a rectangular hyperbola function,
indicating a KD-controlled curve. The brown
curve (right side) describes the scenario
where CBP is higher than the KD. The shape
of that curve is governed by a constant
binding partner concentration (CBPC)
dominated expression, therefore it is a
CBPC-controlled curve. (For interpretation of
the references to colour in this figure legend,
the reader is referred to the Web version of
this article.)

6
A. Vaish et al. Analytical Biochemistry 609 (2020) 113974

monoclonal antibody containing human Fc, which binds to the anti- membrane were incubated for >16 hours (overnight) at room temper­
human Fc coated beads. During FACS analysis, the same fluorescently ature. The membrane-free antibody was separated from the antibody-
labeled anti-target monoclonal antibody must be applied to both the membrane complexes and antibody-free membrane by centrifugation
anti-huFc coated beads and target-bearing cells in the same condition at at 2800xg for 15 minutes. The supernatant was then filtered through 0.2
the same time. Recognized by the same fluorescently labeled anti-target μm 24-well filter plate. The free Ab concentrations in the filtered su­
monoclonal antibody, antibody binding capacity (ABC), which repre­ pernatants were quantified on the KinExA by passing each solution over
sents number of the target receptor/cell, can be quantified with standard the goat-anti-huFc-coated beads, followed by measuring the bead-bound
curve derived from the anti-huFc coated beads. Histogram channel (geo- antibody with fluorescent (Alexa Fluor 647) labeled goat anti-huIgG (H
mean of fluorescence) values corresponding to the specified level of anti- + L) antibody (Jackson Immuno Research). The equilibrium dissociation
human Fc for the bead populations are recorded in a QuickCal® tem­ constant (KD) was obtained by global fitting of the four titration curves
plate for calculation of ABC values (http://www.bangslabs.com/ using the n-curve “Equilibrium, whole cell” model provided in KinExA™
products/flow-cytometry/fluorescence-quantitation). Noteworthy, this Pro software (Sapidyne Instruments Inc., Boise ID). The KinExA Pro
QuickCal® template is designated to the particular QSC kit with the software provides a choice of two equilibrium analysis models, standard
specific lot # as indicated in each template. “Equilibrium” and “Equilibrium, Whole Cell”. The “Equilibrium” model
is applicable to a soluble system in which it has been shown that the
4.2. CD20 membrane preparation using cell disruptor (constant systems) KinExA signal is proportional to the sum of the free binding sites present
in mixture of unbound, singly bound, and doubly bound bivalent anti­
To generate consistent supply of target membrane protein for affinity body [15]. In the case of cells, either intact whole cells or cell membrane
determination, we collected several batches of total 59 billion cells that fragments, all singly and doubly bound Ab is removed by centrifugation
express CD20 between 5e5 to 1e6/cell, averaged at 7.8e5 CD20/cell. On and/or filtration, and only the doubly free Ab is detected. Choosing a
the day of the membrane preparation, the cell pellets (WIL2-S (CD20) model appropriate for the experimental data is important to prevent
cells, total 5.9e10, ~80 g) were weighed and resuspended (approxi­ significant errors in the estimated parameters. In addition, the whole cell
mately 20% or less) in Hypotonic Buffer (10 mM HEPES, pH 7.4, 1 mM analysis provides values (and confidence intervals) for the KD and
MgCl2, 1.8 mM CaCl2) with Roche protease inhibitors (4 tablets/50 mL) expression level (EL – titrant molecules per cell).
and kept on ice/water. One thousand six hundred (1600) mL of the cell
suspension was passed through Constant Cell Disruption Systems with Disclosure of potential conflicts of interest
the disruption pressure 15 psi. Samples were transferred into Beckman
Coulter Ti45 centrifuge tubes and centrifuged at 35,000 rpm Amit Vaish, Peter Grandsard, and Qing Chen are employees of and
(~96000xg) for 20 min at 4 ◦ C. After carefully removing the superna­ own stock of Amgen, Inc. Joanne Ho and Helen McBride are former
tants, the membrane pellets were resuspended in ~50 mL of Membrane employees of Amgen, Inc. No other potential conflicts of interest were
Buffer (10 mM HEPES, pH 7.4, 1 mM MgCl2, 1.8 mM CaCl2, 10% su­ disclosed.
crose) with Roche protease inhibitors (1 tablet/20 mL) and homoge­
nized with 25 strokes using a motor driven homogenizer on ice/water. CRediT authorship contribution statement
Total membrane protein concentration was determined with Bio-Rad DC
Protein Assay Kit (Bio-Rad). Amit Vaish: wrote the manuscript.: analyzed the data. Joanne S.
Lin: performed the experiments, analyzed the data. Helen J. McBride:
4.3. Determination of the equilibrium dissociation constant (KD) of Writing - original draft, critically reviewed and helped writing the
Rituximab binding with CD20 membrane using KinExA® technology manuscript. Peter J. Grandsard: Writing - original draft, critically
reviewed and helped writing the manuscript. Qing Chen: wrote the
UltraLink Biosupport (Thermo Fisher Scientific cat# 53,110) was manuscript, performed the experiments, analyzed the data.
coated with goat-anti-huFc (Jackson Immuno Research) in 50 mM
NaCO3, pH 9.6, followed by blocking with 10 mg/mL BSA in 1 M Tris- Acknowledgments
HCl, pH 8.0. To set up the binding analysis, 10 pM, 30 pM, 80 pM, and
200 pM of rituximab was incubated with titrated CD20 membrane We thank Larry Daugherty for assistance with flow cytometry and
fragments in assay buffer containing 0.1 mg/mL BSA, Roche Protease Tom Glass from Sapidyne for providing insight on KinExA technology
Inhibitor Cocktail (EDTA free), and PBS (without Ca2+, Mg2+). Samples and specific binding analysis of cell membrane targets.
containing the mixture of Rituximab and CD20-expressing cell

Appendix

√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
([Ab]0 − [Ag]0 − KD ) + ([Ab]0 − [Ag]0 − KD )2 + 4[Ab]0 KD
Free Ab (%) = (A1)
2 [Ab]0

Eq (A1) can be rearranged as:

( ) √̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
( )2 ̅
[Ag]0 KD [Ag]0 KD KD
1− [Ab]0
− [Ab]0
+ 1 − [Ab] − [Ab] + 4 [Ab]
0 0 0
Free Ab (%) = (A2)
2
( )
Assuming: X = 1− [Ag]0
[Ab]0
− KD
[Ab]0
KD
and C = 4 [Ab]
0

√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
X + X2 + C
Free Ab (%) = (A3)
2

7
A. Vaish et al. Analytical Biochemistry 609 (2020) 113974

Considering the absolute value of X, |X|, in the square root, and rearranging eq (A3):
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅
X + |X| 1 + |X|C2
Free Ab (%) = (A4)
2
Performing Binomial series expansion of (1 + |X|C2 )1/2 in eq (A4):
[
C C2
X + |X| 1 + 2|X| 2 −
8|X|4
+…
Free Ab (%) = (A5)
2
This expansion is valid for. C
|X|2
<1
Rearranging eq (A5) and ignoring the higher order terms:
X |X| C
Free Ab (%) = + + (A6)
2 2 4|X|
In eq (A6), when [Ab]0 < KD, X would a negative number, whereas |X| is a positive number, which would transform eq (A6) as:
C
Free Ab (%) = (A7)
4|X|
After substituting values of C and |X| in eq (A7):
KD
Free Ab (%) = (A8)
|{[Ab]0 − [Ag]0 − KD }|

For [Ab]0 ≪ KD, eq (A8) can be written as:

KD
Free Ab (%) ≈ (A9)
[Ag]0 + KD

whereas, when [Ab]0 > KD, X is a positive number for [Ab]0 > [Ag]0 and Free Ab (%) is expressed as:
C
Free Ab (%) = X + (A10)
4|X|
Substituting value of C and |X| in the expression:
{[Ab]0 − [Ag]0 − KD } KD
Free Ab (%) = + (A11)
[Ab]0 {[Ab]0 − [Ag]0 − KD }

For [Ab]0 ≫ KD, eq (A11) can be written as:

[Ab]0 − [Ag]0 KD
Free Ab (%) ≈ + (A12)
[Ab]0 [Ab]0 − [Ag]0

For a very narrow concentration regime when [Ab]0 < [Ag]0 , Free Ab (%) expression will not have X component (X is a negative number and |X| is a
positive number, for [Ab]0 < [Ag]0 which will cancel out each other), as a result eq (A12) Free Ab (%) will be for [Ab]0 < [Ag]0 as follows:
KD
Free Ab (%) ≈ (A13)
[Ab]0 − [Ag]0

In summary, for [Ab]0 > KD, Free Ab (%) is primarily expressed by eq (A12) except for a very narrow range [Ab]0 < [Ag]0 where it can be expressed by
eq (A13).

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