Biomedical Engineering Reference
In-Depth Information
engineered cells can provide a consistent reagent over time. Evaluation of cells
(from multiple vendors, PBMC from apheresis of multiple subjects, etc.) is
important to select ones with homogeneous and high surface Ag expression. The
use of cells that express intracellular Ag is not recommended. Ab drug binding to
intracellular Ag depends on the degree of cell membrane permeability; binding
variability could be high. Antigen shedding and/or modulation (internalization)
needs to be examined. Cells that shed Ag may be used if Ag turnover rate is
minimized during staining. In general, fresh cells are more appropriate for PK
analysis because they should best maintain the native target antigen conformation. If
fixed or lyophilized cells are used, comparability of their binding to drugs in
comparison to the fresh cells needs to be demonstrated. Some antigenic epitopes
may change due to fixation or lyophilization.
A relatively homogeneous Ag expression profile is preferred. This is evident by a
narrow symmetrical bell-shaped peak in the histogram (MFI versus cell number)
demonstrating that the data are normally distributed where MFI mean and MFI
median values are similar. Avery broad peak indicates a wide range of Ag expression
among cells, which could cause variability in MFI, resulting in poor assay reprodu-
cibility. Appearance of a tail or a shoulder and/or a double peak could also impact data
analysis. Subcloning of cells may be necessary to select for a homogeneous and high-
target Ag expressing cell population.
Unlike MFI mean, MFI median is less influenced by heterogeneous Ag expression
and therefore is the preferred readout for PK assessment.
The presence of excess Ag for drug quantification is essential. This can be achieved
by careful selection of high Ag-expressing cells and/or use of excess cell number.
High level of surface antigen expression is preferred, which allows drug quantification
across a broad dynamic range. It is recommended that signal/background (signal in
the presence of drug/signal without drug) across the standard curve range should be at
least 2.5-fold.
11.2.2.2 Stable Target Ag Expression
It is essential to select cells with stable
target Ag expression and with minimal shedding and/or modulation upon drug
binding. This should be evaluated over time and through a number of cell
passages; the use of cells with sustained consistent expression is recommended.
For engineered cells, it is preferable to use cells with stable expression, instead of
transient expression. Chinese Hamster Cells (CHO) or mouse EL4 cells are typical
parental cells that can be used to generate target Ag expressing cells.
11.2.3 Cell Processing, Production, and Maintenance
Good aseptic tissue culture technique is critical. General practices of cell processing,
production, and maintenance are described in detail in Ref. [1].
11.2.3.1 Primary cells
Primary cells are generally available in limited quantity.
Some cell types, such as fibroblasts, may be expandable up to certain passages while
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