2014 NJ-Transfer of potency bioassays GLP.indd

Transfer of potency bioassays to a GLP environment, using ADCC reporter bioassays as a case study
Jamil Hantash and Tonya Felix - Tandem Labs, Laboratory Corporation of America® Holdings
Zhi-Jie Jey Cheng, Denise Garvin, Aileen Paguio, Richard Moravec, Teresa Surowy, Mei Cong and Frank Fan - Promega
Introduction
Results
Functional, MOA-based biological assays are required for lot release
and stability testing in biologics drug development. Classic assays
used to measure the Fc effector function for the therapeutic antibody
in antibody-dependent cell-mediated cytotoxicity (ADCC) requires
primary effector cells. These assays are not quantitative, have poor assay
reproducibility, and are challenging to transfer.
In an assay qualification study, the assay demonstrated all required performance
characteristics (repeatability, precision, accuracy, and linearity) as a potency
bioassay for lot release. Both ADCC reporter assays, V and F variant, were able to
measure antibody potencies for multiple therapeutic antibodies known to have
ADCC expressed as MOA, such as rituximab, trastuzumab, and cetuximab.
FIGURE 3
When compared to a classic ADCC assay using PBMCs from homozygous 158VV donors,
the V variant ADCC reporter assay provides equivalent ADCC biological activity ranking
for a panel of glyco-modified trastuzumab mixtures.
The availability of a pair of ADCC reporter bioassays enables the differentiation of
antibody Fc effector function for therapeutic antibodies via FcgRIIIa receptors V158
and F158 variant.
FIGURE 4
Both ADCC reporter assays, V and F variant, measured antibody potencies for multiple
therapeutic antibodies known to have ADCC expressed as MOA, such as rituximab,
trastuzumab, and cetuximab. The availability of a pair of ADCC reporter bioassays enabled
the differentiation of antibody Fc effector function for therapeutic antibodies via FcgRIIIa
receptors V158 and F158 variant. The difference in antibody potency demonstrated in the
two ADCC reporter assays appropriately reflected the differences in IgG binding affinities
for FcgRIIIa receptors V and F variant and correlated with the antibody efficacy in vivo.
FIGURE 6
The ADCC reporter bioassays can work side by side with other potency bioassays for nonADCC MOA-based blocking antibodies such as anti-EGFR antibody panitumumab and
TNFα blocker. When the MOA-based bioassays measure the blocking activity via antibody
Fab domain for panitumumab or infliximab, the ADCC reporter bioassays evaluate the Fc
effector activities for same antibody.
The difference in antibody potency demonstrated in the two ADCC reporter assays
appropriately reflects the differences in IgG binding affinities for FcgRIIIa receptors
V and F variant, and correlates with the antibody efficacy in vivo.
In addition, our work demonstrated that the ADCC reporter bioassays can
work side-by-side with other potency bioassays for non-ADCC MOA-based
blocking antibodies such as anti-EGFR antibody panitumumab and TNFγ blocker
adalimumab.
When the MOA-based bioassays measure the blocking activity via antibody Fab
domain for panitumumab or adalimumab, the ADCC reporter bioassays were able
to evaluate the Fc effector activities for the same antibody.
FIGURE 2
The engineered Jurkat effector cells were further developed in frozen, thaw-and-use
format to reduce handling time, minimize assay variability and facilitate assay transfer.
■
■
A series of trastuzumab glycosylation blend mixtures were prepared by blending
PNGase F – treated (deglycosylated) and untreated trastuzumab (N-glycosylated).
The antibody samples were tested with SK-BR-3 target cells, using untreated
antibody as the 100% activity reference.
TABLE 1
In an assay qualification study, the assay demonstrated the required performance
characteristics (repeatability, precision, accuracy, and linearity) as a potency bioassay for
lot release
FIGURE 1
Two engineered effector cell lines were generated in Jurkat T-cells which
stably expressed a NFAT-RE driven luciferase reporter and either FcγRIIIa/
V158 or FcγRIIIa/F158 polymorphism variant
Accuracy
Method
To quantitatively measure Fc effector function for therapeutic antibodies,
two reporter-based ADCC assays were developed by Promega. For this,
two engineered effector cell lines were generated in Jurkat T-cells which
stably express a NFAT-RE driven luciferase reporter and either FcγRIIIa/V158
or FcγRIIIa/F158 polymorphism variant. The engineered Jurkat effector cells
were further developed in frozen, thaw-and-use format to reduce handling
time, minimize assay variability, and facilitate assay transfer. When compared
to a classic ADCC assay using PBMCs from homozygous 158VV donors, the
V variant ADCC reporter assay provided equivalent ADCC biological activity
ranking for a panel of glyco-modified trastuzumab mixtures.
Expected Relative
Potency (%)
Recovery (%)
150
95.7
125
97.8
75
97.6
50
98.4
25
94.5
Repeatability CV (%)
6.1
Precision CV (%)
9.8
Linearity (r2)
0.995
Covance is the drug development business of Laboratory Corporation of America® Holdings (LabCorp®). Content of this material was
developed by scientists who at the time were affiliated with LabCorp Clinical Trials or Tandem Labs, now part of Covance.
Conclusion
The potency differences as reported by classic ADCC cytotoxicity assays using PBMCs
from donors with V and F alleles.
FIGURE 5
The ADCC reporter bioassays can work side-by-side with other potency bioassays for nonADCC MOA-based blocking antibodies such as anti-EGFR antibody panitumumab and
TNFα blocker. When the MOA-based bioassays measure the blocking activity via antibody
Fab domain for panitumumab or infliximab, the ADCC reporter bioassays evaluate the Fc
effector activities for same antibody.
In summary, the pair of V- and F-variant ADCC reporter assays provides
a valuable approach to quantitatively evaluate the Fc effector function
in ADCC for therapeutic antibodies. It can serve as a valuable tool for
biosimilar, biobetter and next-generation therapeutic antibody drug
research and development.