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Process Development
Kenneth Hoffman
Cygnus
Technologies
P.O. Box 1018
Wrentham, MA 02093
Tel: 508-769-8739
Fax: 508-883-9913
Email: cygnustec@aol.com
Web: www.cygnustechnologies.com
Process Development
technique dependent. Very high molecular
weight proteins cannot transfer sufficiently
and may remain undetected by WB.
Depending on its electrical charge, a protein
may not be released fully from the gel or
bound efficiently to the membrane during the
transfer step, resulting in underdetection.
Very low molecular weight contaminants can
pass through membrane pores without being
sufficiently adsorbed. Sandwich IA requires
that all contaminants have at least two
antigenic epitopes for detection to take place,
so the assay may fail to detect some lower
molecular weight species. In theory, WB
requires only a single epitope for detection.
Sensitivity. The advantage of WB is that it can
separate and help identify individual HCPs. A
multiple contaminate IA, on the other hand,
cannot identify or quantitate one component
from the next. However, IA is more sensitive
than WB. A typical colorimetric WB can detect
bands only at around 1 ng of protein. More
sensitive methods employing photon detection
techniques such as radioactivity or
chemiluminescence can provide detection at
around 100 pg per band, but the sensitivity of
WB is fundamentally limited by the sample size
that can be analyzed. For example, if a typical
10-mL sample is applied to the gel, and the
band detection sensitivity is 1 ng, the
concentration of analyte present in the sample
must be on the order of 100 ng/mL for detection
to occur. Detection limits for IA typically are
less than 1 ng/mL or about 100 fold more
sensitive than WB. That sensitivity is
demonstrated in Table 3 for the HCPs of an
antibody to Chinese hamster ovaries (CHO).
The same affinity-purified antibody directly
labeled with alkaline phosphatase was used in
both a microplate sandwich IA and the WB.
The IA has a 0.5 ng/mL limit of detection
(LOD). The substrate para-nitrophenyl
phosphate (PNNP) used in the IA whereas the
substrate 5-bromo-4-chloro-3-indolyl
phosphate/nitro blue tetrazolium (BCIP/NBT)
was used in the WB. A preparation of CHO
HCPs, solubilized from whole cells, was
subjected to limiting dilution until neither of the
assays could detect HCP. The IA detected CHO
HCPs to a dilution of 1:156,250 compared with
a WB final dilution of 1:1,250. The sensitivity
of the IA was approximately 125-fold greater
than that of the WB.
Measuring multiples. Although IA is
fundamentally a quantitative and objective
procedure, when the assay is trying to
measure multiple components simultaneously,
IA is not quantitative in the strictest sense.
Method
Strengths
Weaknesses
SDS-PAGE/
Silver stain
Subjective interpretation,
qualitative, complex, and
technique-dependent
HPLC
Western blot
Immunoassay
No resolution of individual
components, antibody may
fail to detect some
contaminants
Immunoassay
Quantitative
Semiquantitative
Sensitivity 1 ppm
Table 3. CHO HCP detection sensitivity. Comparison of immunoassay and Western blot.
Dilution of
CHO HCPs
Immunoassay Results
(ng/mL)
WB Results
(# of bands)
1:10
1:50
1:250
1:1,250
1:6,250
1:31,250
1:156,250
1:781,250
250
250
250
176
34
7.1
1.5
assay LOD
40
28
12
5
0
0
0
0
Process Development
purification of the antiserum against the
HCPs. Affinity purification can improve
both specificity and sensitivity. Specificity is
enhanced by the removal of preexisting
irrelevant antibodies and other nonspecific
factors from the polyclonal serum that could
result in artifactual HCP bands in WB or
falsely elevated HCPs in the IA. We
typically see an increase in sensitivity of
greater than 100-fold for IA when affinity
purified antibodies are used in place of an
IgG antisera fraction. Selective enrichment
of HCP-reactive antibody species allows for
lower assay backgrounds, at the same time
increasing the concentration of HCPreactive antibodies. That affinity enrichment
also improves the condition of antibody
excess in the assay relative to the highconcentration HCPs that could be present in
the test sample. By maintaining a high
concentration of antibodies, assay linearity
and analytical range can be extended.
Affinity purification of a polyclonal
antibody preparation using multiple antigens
is not a trivial undertaking, and careful
thought should be given to the strategy.
Although the objective is to enrich the
concentration of HCP-reactive antibodies,
certain HCP antibody species can be lost
because the HCP affinity absorbent does not
contain those antigens. Comparison of WBs
from raw antiserum with those obtained
from affinity-purified antibody should
indicate whether significant antibody
populations have been lost. A perfect
correlation of bands between the antisera
and the purified antibody is unlikely. The
antisera may show non-HCP bands from
nonspecific binding or other artifacts.
Affinity-purified antibody can yield
additional bands over the raw antiserum
because of the selective enrichment and
sensitivity improvement. Multiple WB
bands can be somewhat subjective to count,
but as a general rule we are content with
affinity purification when fewer than 5% of
the raw antiserum HCP bands are missing
afterward. Alternatively, the use of twodimensional gels can be an even higher
resolving method in determining the
reactivity of an antibody preparation.
Calibration of the IA. Much of the difficulty
and misconceptions surrounding assay
calibration are due to a failure to appreciate
the limitations of multiple analyte HCP IA.
The most common mistake is to solubilize
cell proteins and then perform a protein
assay, such as biuret or bicinchoninic acid
Process Development
(BCA), to establish the concentration units
for the IA. Unfortunately, that approach will
rarely yield an accurate or even close
estimation of HCP levels in the product
because dye-binding protein assays and
HCP IAs measure different things. Not all
the proteins are immunogenic. Some, such
as highly repetitious membrane or structural
constituents, will make up most of the total
protein as measured by the protein assay.
However, the HCP assay contains an
insufficient excess of antibody to quantitate
those very high concentration components.
Another reason that solubilized cell proteins
make unreliable determinants of IA
concentration units is that nonprotein
components in the cell lysate, such as the
detergent used for solubilization and cellderived lipids and peptides, can interfere
with the dye-binding protein assay. The use
of IA units calibrated by a protein assay
from whole cell lysate will show falsely
that the IA is relatively insensitive and
will typically result in a significant
overestimation of HCP contamination in
test samples.
Arbitrary units. Analytical purists would like
to report absolute mass or concentration
units, but the practical limitations to
linearity coupled with the arbitrary selection
of antigens for use in both antibody
generation and assay standards make an
absolute measurement nearly impossible.
HCP results should be reported, instead, by
recognizing the limitations of the method
and delivering assay results in units that
acknowledge those limitations. We like to
use the concentration unit of ng/mL of
immunoreactive host cell protein
equivalents. Such an arbitrary unit implies
the uncertainty of calibration yet still
acknowledges that the methodology is
inherently sensitive, semiquantitative, and
capable of providing useful information on
process control. Arbitrary concentration
units have been accepted for years in clinical
diagnostics for many protein hormones and
tumor markers for which a homogenous,
highly purified, well-characterized standard
does not exist. For example, the human
chorionic gonadotropin (hCG) hormone
used to diagnose pregnancy and some other
conditions is assayed by immunological
methods, but the results are reported in
biological activity units of mIU/mL. Those
biological units are based on a historic
nonimmunological bioassay. Because the
immunoassay measures immunoreactive
Process Development
amount of antibody used as the detection
reagent. Because the upper limit of detection
for the assay is determined fundamentally by
the quantity of antibodies, a reasonably good
estimate of the assay range can be obtained by
knowing the quantity of those two antibodies.
The least detectable concentration is more
difficult to estimate because it depends on
other factors such as antibody affinity
constants, nonspecific binding (NSB), and
attributes of the detection and signal
measurement system. With those variables
reasonably estimated, it is possible to estimate
the lower limit of detection based on the
physics of ligand binding.
By the time that the estimated NSB and
detection system background signal is
subtracted, the resulting sandwich assay
standard curve should give a slope of
approximately one over a concentration range
in which the antibodies remain in excess. In
mathematics, that means the slope has been
determined and the upper and lower
asymptotes of the assay ascertained. With the
previous parameters estimated, an antigen
preparation intended for use as a standard then
can be reasonably assigned a value based on its
actual assay dilutional performance compared
with the theoretical mathematic curve. In our
experience, the mathematical method yields a
concentration value for the standards antigen
that agrees closely with the value obtained by
the affinity isolation method.
Strategies for Contaminate Testing
Reprinted from BIOPHARM, Volume 13, Number 6, pages 38-45, May 2000
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