Biologicals (1996) 24, 351–362

Biological Assays: Their Role in the Development

and Quality Control of Recombinant Biological
Medicinal Products
Anthony R. Mire-Sluis, Terry Gerrard1, Rose Gaines Das2, Ana Padilla3 and
Robin Thorpe
Divisions of Immunobiology and 2Informatics, National Institute for Biological Standards and
Control, Blanche Lane, South Mimms, Potters Bar, Herts., EN6 3QG; 1Division of Cytokine
Biology, Center for Biologics Evaluation and Research, FDA, Bethesda, MD 20892, USA; and
3
WHO Biologicals Unit, World Health Organization, CH-1211 Geneva 27, Switzerland

Introduction establish batch to batch consistency, character-

The advent of recombinant DNA technology and its
application in the pharmaceutical industry has
brought about the rapid growth of biotechnology
companies and a number of biological medicinal
products available or under development.1 Many
different proteins have been produced, such as
hormones2,3 (growth hormone, insulin, calcitonin,
follicle-stimulating hormone), cytokines4–7 (inter-
leukins 1, 2, 3, 4, 6, 10, 11, 12, 13 and 15, colony-
stimulating factors, stem cell factor, epidermal
growth factor, neurotrophic factors, fibroblast
growth factors), clotting factors8,9 (factor VIII and
factor IX), vaccines10,11 (hepatitis B and acellular
pertussis vaccine) monoclonal antibodies12,13 (anti-
myosin, anti-lipid A) and, in some instances, in large
quantities to a clinical grade.
The development of biological materials as
therapeutic agents involves the meticulous assess-
ment of safety, efficacy and quality.14–16 Safety and
efficacy are established through well controlled
toxicity studies and clinical trials.17–20 Biological
assays are also important as indicators of safety as
they can detect aberrant potency and illustrate
efficacy as a direct measure of biological activity.
Quality has to be assesed using a variety of
biological, immunological and physicochemical
analytical techniques.21,22
Assessing the quality of biologicals
The quality of a biological product and the process
of its production is assessed by several criteria
including purity, biological potency, stability
and consistency of production.16,23 In order to
ization of the protein product should be undertaken
during product development to establish analytical
criteria and subsequently during batch manu-
facture.24,25 The assessment of stability of the product
relies on the adequate characterization of the
product prior to and during a stability testing
programme. This involves the thorough investi-
gation of structure, physicochemical properties
and biological activity of the protein and any
contaminants.26,27
Physicochemical analyses exploit the unique
characteristics of a protein, although it is often the
case that each test only provides detailed infor-
mation about a single characteristic (Table 1).
In order to provide an overall evaluation it is
necessary to use a combination of these procedures
since none alone can provide enough information
to satisfactorily characterize a product.28,29 Although
many physicochemical tests are available to char-
acterize the structure of a protein product and the
presence of contaminants, they provide little, if
any, information regarding biological potency. The
complex structure of biological materials has meant
that the relationship between their structure and
biological activity is at best only poorly understood
in the majority of cases.30
Biological potency testing
In the context of this review, which will focus
primarily on therapeutic agents (but not including
vaccines which are associated with their own
unique issues), a bioassay is an analytical procedure
utilizing a biological reporter system (resulting
in a biological/functional response), the purpose
of which is to measure the amount of analyte or

1045–1056/96/040351 + 12 $25.00/0 7 1996 The International Association of Biological Standardization

 352

Table 1. Data provided by physiocochemical analysis of biologicals

Data Provided/Inferred

Procedure Size Charge 1° Structure 2°/3° Structure 4° Structure Purity Potency

HPLC:
Size exclusion +++ − − ++ +++ +++ −
Ion exchange − ++++++ +++ − + +++ −
Hydrophobic interaction + + +++ ++ ++ +++ −
Reverse phase HPLC + + ++++ ++ − ++++ −
Circular dichroism − − − ++++ +++ ++ −
MNR + − ++++ ++++++ − ++ −
Mass Spectrometry ++++++ + ++++++ − − ++++ −
SDS gel electrophoresis:
A. R. Mire-Sluis et al .

Reducing conditions +++ − +++ − − +++ −

Non-reducing conditions − − ++ +++ +++ +++ −
Isoelectric focusing − ++++ +++ + + +++ −
Immunoblotting +++ + ++ +++ +++ − −
Immunoassays − − +/− +/− +/− − −
Receptor binding − − ++ +++ +++ − ++
Bioassays − − +++ ++++ ++++ −/+ ++++++
 Table 2. In-vivo and in-vitro biological assays for biological therapeutics
Biological response Biological response
Protein In-vivo assay quantified In-vitro assay quantified
GM-CSF Bone marrow stimulation Number of leukocytes Stimulation of cell lines: Cellular proliferation
TF-1, MO7e, AML-193
TNF Necrosis of tumours Number of tumours Cytotoxicity on cell lines: Cell survival
L929, KYM-D4
Erythropoietin Stimulate erythrocytes in Number of erythrocytes Stimulation of cell line Cellular proliferation
hypobaric rats TF-1
Epidermal Growth Maturation of mouse Time to eye opening Stimulation of cell lines: Cell proliferation
Factor newborn 3T3, Balb/MK, 4MBr-5
Interferon alpha Treatment of virally Elevation of IFN inducible Antiviral activity on cell Cell survival
infected rats enzymes of MHC antigens lines, e.g.: HEp2, WISH,
MDBK
Interleukin 1 Pyrogenivity in rabbits Temperature Stimulation of cell lines: Cellular proliferation
D10, NOB-1, Monomac Production of cytokines
Bone Morphogenetic Bone callous formation Size of callous Stimulation of cell line Production of alkaline
Protein-2 at injection site W20 phosphate
Growth Hormone Growth of rats Weight Stimulation of cell lines: Cellular proliferation,
Biological assays: role and development

NB2, 3T3 differentiation

Heparin Antiithrombotic activity
in rabbits
Influenza Vaccine Mouse immunogenicity
test
Follicle-stimulating Steelman–Pohley test on
Hormone immature female rats
Size of jugular vein Anticoagulant activity Inhibition of activated
thrombi clotting factors
Levels of anti-flu Single radial diffusion Area of precipitation
antibodies
Ovarian weight Stimulation of sertoli Aromatization of
cells or granulosa cells androgens, levels of
cAMP
353
354 A. R. Mire-Sluis et al .
effective constituent in a biological product, i.e. to
determine its biological potency. This contrasts
with binding assays such as immunoassays and
receptor-ligand methods that do not measure the
ability of a protein to induce a biological response.
The most appropriate method for assessing biologi-
cal potency is to compare the biological activity of
a sample to that of a well characterised potency
reference standard. Where possible, it is preferable
to use a response that reflects the biological role
of the material,26,30,31 although it is not necessary to
attempt to mimic therapeutic activity for a test that
is used to assess quality or efficacy.
Such procedures can be carried out in vivo
or in vitro. The choice of bioassay depends upon
the nature of biological material available for
in vitro tests and whether the biological activity
of the biological is only measurable in whole
animals (Table 2).32–39 It is desirable to develop
in vitro biological tests where possible because
they offer distinct advantages over using live
animals.
Live animal experiments suffer from the major
problem of animal-to-animal variation. In order
for an in vivo assay to provide valid estimates of
biological potency, a large number of animals are
required to account for this variation. A great deal
of care and expense is required to decrease any
variation through breeding, housing and feeding
of animals. A balance must also be maintained
between the large number of animals that could
be used to provide several data points for potency
estimates and humane, ethical and economic
pressures to reduce the use of laboratory animals for
assays.
It can be argued that testing in vivo provides
biological potency tests more relevant to the
clinical use of biologicals because a ‘‘whole body’’
approach takes into account bioavailability, serum
half life, toxicities etc. However, this argument is
incorrect because biological assays are not intended
to mimic the biological activity of a product in
the clinical situation. Bioassays used for quality
can illustrate the batch-to-batch consistency of
biological potency of a product as well as defining
the actual potency of each lot of product.
The amount of product required to provide the
optimal therapeutic biological activity in humans—
the therapeutic dose—is determined by clinical
trials. This issue is extremely important as the
biological potency measured for quality purposes
and therapeutic dose are very often linked invalidly
as they are two totally different issues.
Attempts to avoid the requirement for live
animals testing has led to the production of many
different formats for the in-vitro estimation of
biological potency for a wide range of biological
products (Table 2). The use of cells derived from
blood, bone marrow or tissues allows for many
individual estimates of potency with less effort in
comparison to that required to provide a statisti-
cally valid potency for in-vivo tests. However, cells
derived in this manner are still prone to donor
variation and often require careful preparation
procedures. The development of continuously grow-
ing cell lines36 has greatly reduced experimental
variation because cell lines are single clones of cells
that are available in large numbers suitable for
multiple estimates of potency. Also, they do not
require the extensive purification needed for cells
derived directly from in-vivo sources.
Although cell line-based bioassays have distinct
advantages over other biological assays they are
prone to problems that need addressing before
reliable estimates of the levels of any biological
can be derived. The major problem associated with
such cell lines is that they are not specific (e.g.
for a single cytokine36,40). The use of murine lines
increases specificity in some cases as they may not
respond to proteins that are species restricted in
their activity.
The recent development of receptor-transduced
cell lines has produced several more specific cell
lines, but careful maintenance of such lines is
required as the expression of the transduced
receptor is rarely stable and can be lost during
passage. However, even these lines can still respond
to other proteins. It is difficult to find a parent line
that does not respond in one way or another to more
than one biological substance; those that do not
respond may not have the biochemical pathways
necessary to induce biological responsiveness.
Normally, specificity is not a problem for
purified rDNA-derived products. However, should
confirmation of specificity be required, a simple
solution is to include a specific neutralizing
antibody to the protein under investigation with
any test sample and compare the response as
measured in the bioassay of the sample incubated
with and without antibody. The difference in
response should be due to the protein to which
the antibody has been raised and highlights the
response induced by that molecule from any others
contained in the sample.40
Continuously growing cell lines can change their
responsiveness if cultured for long periods and
 Biological assays: role and development 355

therefore cell lines should normally be cultured 34 000

for 3–6 months and then replaced with an early
32 000
passage of cells. Mycoplasma contamination can
also drastically affect the response of cell line-based 30 000
bioassays and it is necessary to check for con-
28 000
tamination at regular intervals. Many bioassay-
associated phenomena such as change or loss 26 000
of responsivenes or lack of tritiated thymidine
24 000
incorporation can be traced to mycoplasma con-
tamination. 22 000
In addition, different batches of fetal calf serum 20 000
used to maintain cultures can greatly affect the

cpm
performance of bioassays and should be carefully 18 000
screened prior to use. Some batches of sera can 16 000
provide excellent maintenance of cell lines (i.e. cells
grow rapidly), but result in poor bioassays with high 14 000
backgrounds or low stimulation indices. Screening 12 000
of sera should include both performance in cell
maintenance and in bioassays. 10 000

The design and analysis of cell-based in-vitro
bioassays
The assessment of the quality and stability of a
product relies on the use of accurate and reliable
test procedures. Of all the tests available for the
analysis of biological products, the bioassay is often
the most variable since it requires the use of live
materials (either animals or cell lines). All analyti-
cal procedures require validation if they are to be of
a suitable reliability and particular attention must
be paid to the validation of bioassays due to their
inherent variability.
The design of a bioassay is not straight forward
since many factors can affect the outcome of the
assay. Bioassay results depend inter alia on the
sample, the reference standard used, the various
reagents, the design and conduct of the assay
procedure and the numerial analysis of the data.41–52
Therefore, during the design of bioassays, efforts
must be made to insure that the bioassay includes,
as far as possible, conditions that test assay validity.
Design of bioassays
There are several approaches that can be adopted
to reduce the variation inherent to bioassays.
The same principles required to reduce animal-
to-animal variation apply to in-vitro cell-based
bioassays, where the growth media, culture con-
ditions and execution of the assay must all be
carefully controlled. It is extremely important to
reduce variation in a bioassay by carrying out
strict maintenance regimes for the cells used in
8000
6000
4000
2000
0
10 100 1000 10 000 100 000
[Sample] (reciprocal dilution)
Figure 1. An illustration of a parallel line bioassay. An
example of a parallel line bioassay where an unknown
sample (w) is titrated through a dilution series and
compared to the dilution series of a reference standard
(W). The biological readout of the assay is shown as the
amount of radiolabelled thymidine incorporated into the
DNA of a proliferating cell line and ilustrated as counts
per minute cpm. The more dilute the sample, the less
proliferation is induced and this results in lower cpm.
the bioassay. Timing of feeding, cell numbers and
materials used should all be kept consistent. One
should also attempt, during incubations for both
cell maintenance and bioassay, to ensure a stable
environment of temperature, humidity and CO2/O2.
Fluctuations due to the opening and closing of
incubators must be avoided and it is recommended
that separate incubators are used for cell mainten-
ance and bioassay. Incubators specifically designed
to reduce variation in internal environment should
be used.
Before a bioassay is carried out, it is vital that the
equipment used to execute the assay, such as liquid
pipettors, is correctly calibrated—preferably prior
to each assay.
356 A. R. Mire-Sluis et al .
Of the various formats available for in-vitro
bioassay, formats based on a 96-well microtitre plate
are rapidly becoming the most popular. Concomit-
tant with this is the development of equipment
designed specifically for the automation of micro-
titre plate based bioassays. However, the wide-
spread use of microtitre plates for in-vitro bioassays
requires certain care in the layout of the plate.
For instance, it has been shown that the wells on
the edges of microtitre plates are subject to greater
variation than internal wells. It is therefore
recommended that the outer wells of microtitre
plates are not used for estimates of biological
potency, but must be treated in a similar manner
to the rest of the plate, but without inclusion of
cells. The inclusion of coded duplicates during assay
design must be paralleled in practice with several
internal duplicates included in the assay.
Although internal plate variations are difficult
to avoid, it is possible to design the layout of
successive plates within an assay to minimise such
effects. Randomization of sample position appears to
be the most efficient method to achieve this. If
samples are rotated on successive plates, as well as
shifting the order of neighbouring samples, many
plate effects can be minimized.
The basic format of all biological assays involves
the comparison of an unknown sample to a reference
preparation. The most appropriate format to carry
this out is by creating a dilution series of each
preparation. The use of single-point assays is
not suitable due to the lack of information they
provide about the performance of the bioassay and
samples. A dilution series will result in a standard
dose–response curve of the reference preparation to
which the dose–response curve of an unknown
preparation can be compared (Fig. 1). A great deal
of information about the validity of the bioassay can
be derived from the statistical evaluation of a
comparison between these dose response curves.
Validating bioassays
The fundamental condition for assay validity is
the condition of similarity of sample and reference
standard; that is, the dose–response relations
(i.e. slope, asymtopes etc.) for the sample and the
reference standard should be identical.53
During assay development substantial infor-
mation about dose–response curves should be
collected in order to select an optimal dose range for
potency estimation assays. After such data are
available, analysis in terms of log doses is often
found preferable to analysis in terms of absolute
units. The optimal assay range is often chosen in
a linear (or linear under suitable transformation)
part of the log dose–response relation. In such
a situation, the condition of similarity becomes a
condition that the log dose–response line for the
sample should be parallel to that for the reference
standard, i.e. a parallel line assay (Fig. 1). Provided
at least three or ideally more doses of each
preparation are included in the assay, the con-
ditions of linearity and parallelism of the log
dose–response lines can be tested in the individual
assay. Moreover, the slope of the line or other
characteristics of the responses may also provide
information about conformity or otherwise with
the previously determined complete dose–response
relation.
Various assumptions about the statistical nature
of the assay response data must be satisfied if
estimates derived from such analyses, and the tests
for the conditions of linearity and parallelism
given by such analysis, are to be valid. These
criteria must be experimentally determined and the
bioassay design amended to take into account how
such ‘‘variations’’ might affect the bioassay results.
Although it may not be possible to exhaustively test
these assumptions about the nature of the response
data within individual assays, each assay should
include appropriate replication to permit at least
some testing.
First, one must assume that the ‘‘experimental
units’’ providing the response represent a random
selection from a defined population of such units. In
microtitre plates for example, the response unit is
often a set of triplicate wells. It must be assumed
that any three wells positioned anywhere on the
plate (or between different plates) are identical in
nature. However, exceptionally careful definition is
required both for the ‘‘units’’ which are responding
and for the ‘‘treatment’’ which they are receiving.
In microtitre plates the position of the well is
critical in the definition of the unit. Wells in some
rows or columns may be consistently different from
others. Results obtained for cells applied earlier to
the plate may be different from those applied later.
Units may differ as a result of a temperature, oxygen
or humidity gradient across the plate. If an assay
extends over several microtitre plates such differ-
ences between wells becomes even greater so these
and other factors must become part of the definition
of the ‘‘experimental unit’’.
It must also be assumed that the responses are
completely determined by the specified doses except
for independent and normally distributed random
 Biological assays: role and development 357

156%

125%
Limits around
the mean Mean stated Fiducial limits
stated potency potency of assay

85%

64%

1 2 3 4 5 6 7 8 9 10 11
Batch number

Figure 2. An example of how statistical limits are applied to biological assays. A biological should be assayed in several
independent bioassays. Each independent assay will provide an estimate of the potency of a sample from a specified
batch of the biological (r). A mean potency (W) for the batch is obtained from the independent estimates for that batch
and fiducial limits about that mean are obtained using the variability among the log potency estimates. The limits
around the stated potency are determined on the basis of required accuracy of biological administered to patients. The
fiducial limits are set to allow for the variation inherent in bioassays and to ensure confidence in the calculated mean
value for any batch.

deviations. This is a particularly critical assumption
and an assumption which it is easy to violate
inadvertently. It can be difficult to ensure that the
treatment applied to the responding units consists
only of the specified dose. It is seldom possible for
all experimental units to be treated simultaneously
which leads inevitably to time differences. Some
doses will be exposed longer than others to
particular conditions before being applied. Some
cell populations will have had a longer time with the
treatment than others; different parts of the assay
will have been carried out at different times
(however slight these may seem). Other treatment
differences may arise if the units treated with some
specified doses on one microtitre plate are more or
less vigourously washed than those treated with
other doses on another plate. It appears to be
especially difficult to ensure absolutely uniform
treatment of the whole of a plate during incubations
over more than a few minutes.
It is also important to show that the variances
of the deviations are homogeneous over the
different doses. The basis for the various tests
of validity of an assay as well as the fiducial
limits (i.e. variation) of the potency estimate is the
deviation of the observed responses from the mean
response.
The fiducial limits of the assay are calculated
from the variation between replicate responses
(Fig. 2). If, however, the responses are not
completely determined by the specified doses,
then this variation cannot be random and indepen-
dent, violating one of the essential statistical
assumptions. For example, if replicate responses
are obtained from neighbouring wells of a micro-
titre plate, then they are likely to have been
influenced by other common factors than just
the common specified dose (the ‘‘plate effects’’
already described). Their deviations from the
dose-determined response are thus correlated (i.e.
not independent), having been influenced by the
presumed common factors. The deviations are
also not random because these factors do not
occur by chance and therefore do not apply equally
to any other responses situated elsewhere in the
plate.
It has been out experience that failing to take
such effects into account leads to underestimation
of the response variability. This in turn can lead to
apparently significant departures from linearity or
parallelism and in others to substantial underesti-
mation of the width of the fiducial limits.
Statistical analysis of bioassays
The statistical analysis of bioassays must incor-
porate not only the estimation of biological potency
but also include analysis that takes into account the
assay validity. Various approaches can be taken
in the statistical analysis of bioassays to take
such eventualities into account. Methods for the
358 A. R. Mire-Sluis et al .
statistical analysis of parallel line assays have
been described.53 In some fields, statistical methods
have been developed which attempt to measure
and account for some of the common factors
(e.g. row and column effects) so that they do not
influence the analysis in misleading ways. It has
been our experience that such factors are not
in general sufficiently consistent or predictable
to make this a profitable approach. Some assay
procedures appear to be more subject to the
influence of ‘‘unexpected common factors’’ than
others and there are exceptions to any broad
generalizations.54
An approach which can provide valuable infor-
mation concerning the precision of the whole
assay procedure is the inclusion in assays of a
coded duplicate.49,50,54 For this, two ‘‘identical’’
preparations which are considered by the analyst to
be either a sample and a reference standard, or two
independent and different samples are compared
in the same assay. If the relative activity of these
two preparations differs consistently from 1 then
it is likely that some phenomenon is affecting
one preparation differently than the other. If the
deviation of the relative activity from 1 is greater
than that predicted on the basis of the fiducial
limits, then it is likely that the response variability
has been underestimated.
Clearly the most reliable measure of assay
precision is that obtained by direct calculation
using potency estimates from independently re-
peated assays of the same pairs of preparations,
although this may presuppose that the condition of
similarity is satisfied.
Biological standardization
The specific activity of various recombinant
forms of a biological made by different manu-
facturers can vary greatly and therefore it is not
possible to quantitate the biological activity of
rDNA derived products by mass.41,42 Because 1 mg
of one manufacturer’s material does not have
the same biological activity as 1 mg of another
manufacturer’s material, the use of mass units is not
appropriate. As discussed, biological systems are
subject to change and the biological responses may
be affected by a number of factors which, even
if known, may not be under the control of the
operator. EC50 values in particular may shift as the
result of changes in cell lines, reagents and assay
conditions.
The assay of biological products, in common with
other measurements, is essentially comparative and
thus requires some fixed standard.43,44 Although
there are instances of attempts to produce ‘‘stan-
dard assay systems’’, beginning with the use of
animals in which the biological effect of different
products could be observed, these have been largely
unsucessful.45,46 In order to estimate the potency
of a succession of unknown samples, the samples
must be compared to a consistent reference
standard. In-house reference standards, usually
consisting of a single well-characterized batch of the
biological product, are generally used for the day to
day potency estimates of a manufactured product.
However, if the potencies of different preparations
of the same protein are to be compared by different
operators using different bioassays worldwide,
the need for international reference standards and
international unitages is fundamental. The use of
various individually derived units and conversion
factors causes confusion.
The World Health Organization (WHO) has an
active and long-standing programme for the inter-
national standardization of biological materials.
International potency standards for a large range of
biological materials are available world-wide for the
standardization of biological assays to internation-
ally recognised reference preparations with defined
International potency units. There are therapeutic
products that are soluble receptors or antagonists
for proteins and in such cases, these materials are
standardised against their equivalent primary
ligand.
Regulatory aspects of quality assessment
Guidelines have been prepared in both the U.S.A.
and E.C.23,55,56 containing details for the assessment
of the potency and stability of rDNA derived
products. Protocols for the design and analysis of
bioassays are also included in the European57,58 and
U.S. Pharmacopoeias.59 It is implicit in all texts that
biological assays are a vital part of the assessment
of biological products. Although exact formats of
bioassays are not discussed, generalized issues have
been included. However, the statistical evaluation
of bioassays are covered in the pharmacopoeias
to some depth. It should be noted that it is
recommended that in-house reference preparations
(or working standards) are standardised to the
WHO International standard when available—if
not, it is recommended to use a National Institute
for Biological Standards and Control (NIBSC)
interim standard.58,60
 Biological assays: role and development 359

Alternatives to biological assays aspects of the characteristics of biologicals. The

data produced by these different types of procedures
Receptor binding techniques have been developed
complement each other to provide a spectrum of
in an attempt to correlate binding affinities to
information on the substance and different batches
biological activity. The value of such assays is
of product. Although some of this may overlap,
limited by the quality of labelled ligand (and its
the different assay types provide data which relate
consistency), the receptor system used and the
to different properties of the molecule in question.
reliability of the data provided.61–63 It must be
There is a great deal of debate over the necessity
remembered that there exist naturally occurring
for many of the tests required by control authorities
and rDNA-derived protein antagonists that bind
and in particular the need for a bioassay for
to receptors with the same (or increased) affinity
rDNA-derived materials. However, although one
as the parent protein but do not induce any
can argue that the technology used to manufacture
biological responses.64–66 Mutated proteins with
these materials is no longer so novel as to warrant
altered (or no) biological activity can have identical
such caution, it must be stressed that it is the
receptor binding patterns to the unmodified
biology of the molecules being produced that is
molecule.67,68 Some altered molecules can exhibit
relatively poorly understood, not the manner in
increased affinity associated with decreased
which they have been produced. The majority of
biological activity or vice versa.69,70 Differences in
rDNA-derived proteins, e.g. the cytokines, are only
glycosylation can also affect both receptor-binding
available for research for short periods of time
characteristics and biological activity of proteins,
before being tested in the clinics. It is impossible in
again with an unpredictable relationship.71,72 There-
that time period to predict from even the most
fore, receptor-binding affinity is not necessarily a
sophisticated analytical technology available, how
good correlate of biological activity.30 Although
subtle changes in protein structure could affect
receptor-binding assays may be useful in monitoring
the biological potency of these molecules.30 It has
batch to batch consistency, accurate assessment of
taken decades of research on biologicals such
biological potency necessitates the development of
as growth hormone and insulin to reach a stage
correctly executed, analysed and standardised
where the biological activity of these proteins can
bioassays.26,73,74
be correlated with physicochemical analysis75,76 with
Immunoassays are another alternative to assay
reasonable assurance. For more complex thera-
biological products. The major drawback with
peutic biologicals showing multifactorial activity,
immunoassays is that they can detect both biologi-
physicochemical analysis may never be able to
cally active and inactive molecules and cannot be
replace bioassays unless the pleiotropic biological
used as an indicator of the biological potency of
activities of these products are fully understood.
a product.1 In addition, although it is generally
Until that time, biological assays are the only valid
accepted that immunoassays are more reproducible
and reliable analytical method for assessing the
and accurate than bioassays, there have been
biological potency of these biological products.
several studies to show that well controlled
bioassays can be as reproducible and accurate
(if not more so) as immunoassays.41,42
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Received 29 April 1996;
accepted for publication 20 May 1996