Biomedical Engineering Reference
In-Depth Information
(a)
(b)
125
100
100
Ligand affinity purified antibody
Irrelevant purified antibody
80
75
60
50
50
40
25
Rabbit sera 1
20
Rabbit sera 2
Prebleed rabbit sera
0
0
512
256
128
64
32
16
8
4
0
20
40
60
80
100
Dilution factor
ยต
g/mL antibody
FIGURE 10.2
Evaluation of matrix interferences and specificity of positive control(s) used
as sera (a) or ligand affinity purified (b).
whether the use of a particular cell line is feasible, although often times the most
relevant cell line for immunogenicity testing is also the one used for the biotherapeutic
drug potency assay. Neutralizing antibody assays typically measure a decrease in
signal, but if the mode of action of the biotherapeutic results in a decrease in signal,
neutralization of this activity would then be seen as an increase in signal [1].
The success or failure of each analytical run can be directly related to the
characteristics of the cells in use. Therefore, the influence of critical cellular attributes
needs to be fully examined during method optimization. This should include studying
culture conditions to determine whether changes in ligand or receptor expression are
related to culture conditions, cell passage, the degree of confluence, and changes in
culture media formulation, in particular, the serum content and source, and whether
these changes have any effect on the assay performance [1].
An example of the effect of passage on uptake of labeled drug is included in
Figure 10.3. In this example, monitoring cell passaging (frompassage 6 to 38) and cell
confluency shows that the active uptake and accumulation of labeled drug is higher in
postconfluent cells (Figure 10.3a). However, the percentage inhibition of drug uptake
(Figure 10.3b) and inhibition by a neutralizing positive control (Figure 10.3c) were
not significantly different across a range of cell confluencies or cell passage number as
assessed using one-way ANOVA and Wilcoxon Rank Sum tests.
During assay feasibility studies, other cell line attributes may be associated with
changes in assay performance, such as a decrease in proliferation rate or a change in
cellular morphology. These changes could potentially have a direct influence on assay
performance; at minimum, they should be documented and tracked along with assay
performance. One example of the utility in tracking cellular growth attributes with
assay performance is a suspension cell line that was grown and passaged during a
NAb assay validation. Toward the end of the assay validation, a significant decrease in
assay signal was observed and traced to a change in the cell growth characteristics -
from loosely attached and clustered cells to suspended single cells (data not shown).
These types of growth variations are subtle and sometimes hard to identify; therefore,
it is highly recommended that growth characteristics, cell seeding density, and any
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