SCIENTIFIC REPORT

Biacores SPR technology in a

GMP-regulated environment
K.Olaf Reynhardt and Meena Subramanyam
Biogen Inc., 14 Cambridge Center, MA 02142, USA

Ligand binding assays, developed using Biacores SPR technology, have proven invaluable in the characterization of biopharmaceuticals to determine
consistency of the manufactured product. We have developed and validated
an equilibrium binding assay on the Biacore 2000 instrument for measuring
the apparent binding affinity of a soluble receptor fusion protein to its cognate ligand. The soluble receptor fusion protein (alefacept) has a low affinity
(M range) for its cognate ligand, CD2. In this article, we describe how we
validated a binding assay for product release and stability testing in compliance with ICH guidelines

he release of a pharmaceutical product

for clinical use requires a thorough characterization of its biological activity,
the specific ability or capacity of a drug to produce a defined effect. Biological assays measure
the biological and physiological response to the
drug in vivo in animals or in vitro in cell culture-based systems. In the quality control environment, special attention is given to the
robustness of analytical methods. Needless to
say, animal- and cell-based assays are cumbersome and are frequently unreliable. Receptor
binding assays are a reasonable substitute for
traditional biological potency assays, provided
there is sufficient physicochemical information
about the drug, and a good correlation with
biological activity is demonstrated (receptor
binding affinity may not necessarily correlate
with the potency of the drug) (1). Moreover,
FDA directives require that binding assays are
part of the product release portfolio, if the drug
product is an antibody.
Binding assays that use purified receptors or
membrane preparations perform better than
whole cell assays, are easier to run and can be
easily automated. We have developed a Biacorebased binding assay, using purified components, for routine use in product release.
Alefacept is a bivalent fusion protein comprised
of the first extracellular domain of LFA-3 fused
to the hinge region (CH2 and CH3 domains) of
human IgG1. It binds to CD2 on the surfaces of
immune cells and inhibits the immune response,
particularly of T cells. Due to the low binding

PAGE 12

affinity of alefacept to CD2 (2), we developed a

binding assay using Biacore 2000. Using SPR
technology, low affinity binding interactions
can be monitored in real time without the labeling or washing steps common in other techniques. Recombinant human CD2 is covalently
immobilized to the surface of a CM5 sensor
chip by amine coupling. Duplicate alefacept
reference standards and samples are injected
into the flow cell at 8 dilutions between 12.3
nM and 3000 nM, in a random order, and are
allowed to interact with the immobilized CD2
until equilibrium is established (Figure 1a). The
SPR equilibrium resonance unit values (Req RU)
are determined and plotted against alefacept
concentration (Figure 1b). A nonlinear curve
fitting of the BIAevaluation 3.0 software is used
for data analysis. Results are reported as an
apparent KD value (dissociation affinity constant (M)).
BIACORE BINDING ASSAY VALIDATION

For use in product release the assay was validated according to ICH guidelines (3). No specific
guidelines are currently available for validation
of binding assays. One of the goals of the
Ligand Binding Assay Bioanalytical Focus
Group under the umbrella of AAPS (American
Association of Pharmaceutical Scientists) is to
achieve a pharmaceutical industry consensus
for harmonizing validation of receptor binding
assays. Until such consensus is established, QA
departments most probably will be required to
comply with the current ICH guidelines for
Biacore Journal Number 1 2001

SCIENTIFIC REPORT

RU

Figure 1a

Response

100

A representative set of binding sensor-

60

grams of alefacept binding to CD2.

20

Duplicate alefacept reference standards

-20

and samples are injected into the flow cell

-60
0

50

100

150

200

250

300

350

400

at 8 dilutions between 12.3 nM and 3000

Time s

nM, in a random order, and are allowed to

interact with the immobilized CD2 until

Fig. 1a

equilibrium is established

Req
70
60
50
40
30
20
10
0
-10

Figure 1b
A representative plot of C versus Req. The
0

5e-7

1e-6

1.5e-6

2e-6

2.5e-6

3e-6

3.5e-6

SPR equilibrium resonance unit values

(Req RU) are determined and plotted

Fig. 1b

Alefacept Concentration (M)

assay validation, which include validating assay

(i) precision, (ii) accuracy, (iii) specificity, (iv)
linearity, (v) range and (vi) robustness (4). Some
degree of adaptation of these definitions was
necessary in designing the Biacore alefaceptCD2 binding assay validation protocol.
(i) Precision is the measure of repeatability of
an analytical method under normal operating
conditions. Precision is comprised of repeatability and intermediate precision. Repeatability
refers to the results of the method operating
over a short time interval under the same conditions. The 8 point alefacept reference standard curve was tested over ten days in 5 individual runs (5 sets), in duplicate, on the same
flow cell of the chip. The chips were freshly
coated with CD2 every day. The standard deviation and coefficient of variation (%CV) of the
apparent KD was calculated for each day. Figure
2 shows that the %CV for the individual days
varied between 4 % and 24 %. With the exception of one day when the value was 24%, %CV
values were below 15 % for the individual runs.
Intermediate precision refers to the results from
variations within a single laboratory due to random events such as differences in experimental
periods, analysts or equipment. Two analysts
tested the assay standards over five days, 5 individual runs/day in duplicate, on different chips.
The standard deviation, coefficient of variation
and 95%CI of the apparent KD were calculated
for all 10 runs. The %CV for intermediate
assay precision was 16 %.
(ii) Accuracy is the measure of exactness
Biacore Journal Number 1 2001

against alefacept concentration

of an analytical method, or the closeness of

agreement between the measured value and the
value that is accepted either as a conventional,
true value or an accepted reference value.
Accuracy was established from 50 measurements as a percentage of measured KD value
relative to the accepted true reference value of
0.75 M. A 95 % CI for percent recovery and
SD values were established. As shown in Figure
3, the recovery values for 47 individual standards sets were between 77 % and 128 %. The
recovery value of three of the samples lay outside this range.
(iii) Specificity is the ability to measure, accurately and specifically, the analyte of interest in
the presence of other components that may be
expected to be present in the sample. Modified
(stressed) alefacept samples (aggregated, oxidized, deamidated) were analyzed. The result was
reported as an apparent KD value and as a percent recovery in comparison to a reference standard on the same chip. Alefacept samples containing a certain amount of oxidized material yielded only a 50 % recovery, which indicated that
oxidation altered the binding affinity to CD2.
Aggregated samples had a higher apparent binding affinity with a recovery of 166 % (aggregates evidently generate higher RU values on the
chip and thus give a false high apparent binding
affinity). Deamidation had no significant effect
on the binding affinity between CD2 and alefacept, with the 121 % recovery falling within the
expected accuracy values of the assay. These
results suggested that the assay is able to detect
PAGE 13

50

SCIENTIFIC REPORT

45
40
35

Assay precision: the measure of repeata-

30

bility of an analytical method under nor-

%CV

Figure 2

25

mal operating conditions. The 8 point ale-

20

facept reference standard curve was tes-

15

ted over 10 days in 5 individual runs (5

10

sets), in duplicate, on the same flow cell of

the chip. The %CV for the individual days

0
0

varied between 4 % and 24 %

10

Validation Run
160

Figure 3

140

+2 SD

Assay accuracy: the measure of exact120

seness of agreement between the measured value and the value that is accepted
either as a conventional, true value or an
accepted reference value. Accuracy was

% Recovery

ness of an analytical method, or the clo-

Mean

100
80
60

-2 SD

40

established from 50 measurements as a

percentage of measured KD value relative
to the accepted true reference value of
0.75 M.

20
0
0

differences in binding caused by the presence of

modified alefacept molecules in the sample.
(iv) Linearity is the ability of the method to
give results that are directly proportional to the
analyte concentration within a given range.
Linearity, established from 50 measurements
and expressed as a Chi2 value (the mean square
of the signal noise), describes how well the
results approximate those calculated from the
model used to analyze the data (KA x concentration x Rmax/(KA x concentration x (n+1))).
(v) Range is the (inclusive) interval between
the upper and lower analyte concentrations that
have been demonstrated to be precise, accurate
and linear. Assay range was chosen during the
method development phase. The concentrations
included 8 dilutions in duplicate, between 12.3
nM and 3000 nM, which fully covered the anticipated range of apparent KD values. Validation
of precision, accuracy and linearity also validated the assay range. The average Chi2 value for
the evaluation over 10 days was 7.1, with a
range between 1.9 and 15.9. Taken together
with precision and accuracy, this also confirmed our selection of the assay range.
(vi) Robustness is the capacity of a method
to remain unaffected by small deliberate variations in method parameters. Variations of the
chip coating density were introduced (at or
close to 300 RU, 350 RU, 400 RU, 500 RU,
550 RU and 600 RU). A set of 6 assay standards, in duplicate, was analyzed and the allowable deviation from the target 450 RU coating
density was established.
PAGE 14

10

Validation Run

Finally, system suitability is a validation

parameter that ensures that both methodology
and instrumentation are performing within
expectations. Biacores SPR technology is well
suited for a regulated environment: the Biacore
control software integrates system maintenance
and system check protocols, which need to be
utilized periodically with appropriate record
keeping. Biacore maintains a well-established
training course for new operators.
In summary, Biacore has been satisfactorily
used in a regulated product development/QC
environment. The sensitivity, increased sample
throughput and ease of use of the Biacore system enabled the development of a specific and
highly reproducible assay for a soluble receptor
with low affinity for its cognate ligand.
References
1. Test Procedures and Acceptance Criteria for
Biotechnological/Biological Products. Q6B
Specifications, FDA Docket No. 98D 0374
(August 18, 1999)
2. Majeau, G.R., Whitty, A., Yim, K., Meier,
W. and Hochman, P.S. Low affinity binding of
an LFA-3/IgG1 fusion protein to CD2+ T cells
is independent of cell activation. Cell Adhesion
and Communication 7: 267 (1999)
3. International Conference on
Harmonization, Guideline on Validation of
Analytical Procedures: Methodology, Federal
Register 61: 59 (1996)
4. Bruno, J. Validating Biacore Assays.
BIAjournal 2: 9 (1999)
Biacore Journal Number 1 2001

SCIENTIFIC REPORT

Biacores SPR technology and DNA footprinting

for the discovery and development of new
DNA-targeted therapeutics and reagents
W. David Wilson, Lei Wang, Farial Tanious, Arvind Kumar, David W. Boykin, Carolina Carrasco1 and
Christian Bailly1 Department of Chemistry, Georgia State University, Atlanta, Georgia 30303 USA and
INSERM U-524 et Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret, IRCL, Place
de Verdun, 59045 Lille, France

A number of clinically useful agents against diseases, from cancer to infectious disease, exert their therapeutic action by interacting with cellular DNA
and causing some perturbation in protein-DNA recognition. Discovery of
compounds with enhanced DNA-targeting capability is critical to the development of potential drugs of this type. We describe here the use of a complementary approach involving compound design and synthesis, a combinatorial analysis of binding sites by DNA footprinting methods and high resolution Biacore analysis to discover and characterize new DNA targeting and
interaction modes with increased specificity and affinity

lthough the first draft of the complete

human genome sequence was announced almost one year ago and the genomes of a large number of organisms are now being
sequenced, the use of this vast amount of new
information is just beginning. One of the challenges presented by this array of sequence information is to design new types of compounds that
can readily penetrate the cell nucleus and interact
with a desired target sequence of DNA. Such
compounds would have multiple uses in therapeutics and biotechnology. We have found that
even relatively small molecules can have remarkable good therapeutic effects by targeting DNA
of certain organisms. A prodrug of furamidine,
(DB75 in Figure 1), for example, has recently
completed phase I clinical trials and has activity
against several infectious disease organisms ranging from Pneumocystis carinii to trypanosomes.
The compound is already scheduled to enter two
phase II trials this year. DB75, which is the active component, is known to bind strongly in the
DNA minor groove with selectivity for AT-rich
sequences (1). Its therapeutic action is thought to
Biacore Journal Number 1 2001

come from selective uptake by target cells with

inhibition of one or more DNA-directed enzymes
or control proteins in those cells. These initial
studies demonstrate that the compound readily
enters cells and has low toxicity to humans at
therapeutic doses. The prodrug and other compounds under development are very promising
advances in the treatment of infectious diseases
that affect millions of people worldwide.
Development of additional compounds in
this series offers the potential of an improved
therapeutic ratio as well as possible generation
of additional biological activities against other
organisms or disease cells including cancer cells.
For these reasons derivatives of DB75 are being
synthesized and their interactions with DNA
are being characterized. Synthesis of benzimidazole derivatives and replacement of the phenyl
rings of DB75 with benzimidazole rings is a
high priority since benzimidazoles offer the
potential of expanded DNA base pair recognition (Figure 1). Benzimidazole isomers, unlike
the phenyl rings of DB75, can recognize AT
base pairs through donation of a hydrogen

O
NH2
+
NH2

H2N
+
H2N
N

N
H

H 2N
+
H2N
N

DB293
NH2
H2N +
N

O
N
H

N
H

DB270

H 2N
+ NH
2

DB75

H2N

NH2

H
N

N
H

G-NH2

AN3/T02
Fig. 1

Figure 1
Structures for the symmetric compounds
DB75 and DB270 as well as the nonsymmetric compound DB293 that display the
new dimer DNA recognition mode.
Possible recognition of AT and GC base
pairs by benzimidazole isomers is also illustrated.
PAGE 15