Biomedical Engineering Reference
In-Depth Information
others such as PBMC undergo limited expansion or require cytokines/growth
factors for their expansion, which may change cell phenotype such as surface
antigen expression. Cell passage analysis should be performed to establish limits
within which Ag expression remains high and stable. A master cell bank should be
established and working cell bank generated to support both assay development
and validation studies and in-study/clinical sample testing. Optimal cryopre-
servation and cell thawing procedure should be established with acceptable cell
recovery and viability, and should maintainAgexpression.Freshlythawedcells(in
a single-use aliquot) from the working cell bank may be used directly. Cells may
also be kept in culture after thaw as long as target Ag expression and cell viability
are maintained within the established time period or passage number. Fixed and/or
lyophilized cells may be used once they are well characterized to demonstrate
comparability with the characteristics of fresh cells such as Ag expression and
bindingtodrug.
Some cell types, such peripheral blood mononuclear cells (PBMCs), are not easily
expanded over time without changing cell phenotype. PBMCs can be isolated from
venous blood following two commonly used procedures, Ficoll gradient centrifuga-
tion (commercially available for human PBMC) and CPT cell preparation tubes (BD
Biosciences), following the vendor recommended procedures described in the
product inserts. Apheresis products may also be used. Due to individual donor
variability, PBMC from multiple healthy subjects should be evaluated and cells with
relatively homogeneous high Ag expression selected. Cell banks with frozen PBMC
in single-use aliquots should be established from the chosen PBMC donor. It is not
recommended to refreeze thawed cells.
The example in Figure 11.1 illustrates binding of a polyclonal Ab drug (drug X) in
a dose-dependent manner to PBMC that expresses the target Ag. The Ag expression
profile is shown as histograms andMFI-medianvalues are indicated (data collected on
FACSCalibur
). This particular method is intended for PK analysis in humans.
11.2.3.2 Cell Lines and Engineered Cells
For transformed cell lines, cell growth
and maintenance procedures need to be established including cell feeding and culture
scheme, Ag expression, and cell number, growth, and viability. Generation of amaster
cell bank and working cell banks is necessary to minimize lot-to-lot variability. Easy
maintenance, good growth rate, expandability, and stability (both freeze/thaw and
long-term storage) are additional criteria for selection of a suitable cell line. Amaster
cell bank and working cell bank need to be established for method validation and in-
study/clinical sample testing.
Figure 11.2 illustrates a monoclonal humanized Ab drug (drug Y) binding to
engineered CHO cells expressing the Ag (CHO-Ag). Representative histograms are
shown with MFI median indicated (data collected on FACSCanto
II). This assay is
also intended for PK analysis in humans. Similar to PBMC results, the use of
CHO-Ag also demonstrated dose-dependent binding across the full drug concentra-
tion range. Ag expression on CHO cells is more homogeneous (a very narrow peak in
the histogram) compared to the profile seen with PBMC containing different
subpopulations of lymphocytes and monocytes, as expected.
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