Recombinant Technology
Pharmacokinetics of CAMPATH-1H:
assay development and validation
Peppy Rebello, Geoff Hale
*
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK
Received 25 July 2001; received in revised form 2 November 2001; accepted 2 November 2001
Abstract
CAMPATH-1H is a humanised monoclonal antibody against the CD52 antigen which is being developed for treatment of
chronic lymphocytic leukaemia (CLL), autoimmune disease and prevention of transplant rejection. Measurement of antibody
serum levels is important for optimising dose regimens but difficult owing to the low concentration compared with normal
human IgG. After consideration of various methods, a suitable assay was developed based on indirect immunofluorescence.
Test samples were incubated with target cells (HUT-78, a human T cell line) and the CAMPATH-1H was detected by binding of
a fluorescent-labelled anti-human Ig using a flow cytometer. Robustness of the assay was demonstrated under a range of
experimental conditions. Because of the low affinity of CAMPATH-1H, only a weak signal was seen at low concentrations. The
limit of detection was 0.15 mg/ml and the limit of quantitation was 0.25 mg/ml. Since serum samples were diluted at least 1:2, the
lowest concentration which can be measured in patient serum was 0.5 mg/ml. The overall precision (coefficient of variation)
was F 13% and the overall accuracy (bias) was + 9%. There was a low incidence of false-positive results ( < 2%) in normal or
pre-treatment patient serum. Quantitative recovery was obtained from serum samples spiked with CAMPATH-1H and stored
under a variety of conditions, including being treated at 56 °C for 30 min and frozen and thawed up to four times. This validated
assay is suitable for the measurement of CAMPATH-1H levels in clinical trials and the same principles may be applied to any
other cell-binding monoclonal antibody. D 2002 Elsevier Science B.V. All rights reserved.
Keywords: CAMPATH; CD52; Validation; Immunofluorescence; Pharmacokinetics
1. Introduction
An important part of drug development is the
measurement of pharmacokinetic properties. Under-
standing biodistribution and kinetics of clearance can
be crucial to achieve the optimum efficacy and mini-
mum toxicity. It is essential to have a measurement
technique which is sufficiently accurate and sensitive
and does not suffer interference from other substances
normally found in the samples (typically serum). In
the case of a humanised monoclonal antibody, assay
development is challenging because the antibody,
0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
PII: S 0022-1759(01)00556-7
Abbreviations: ADCC, Antibody dependent cellular cytotox-
icity; BSA, Bovine serum albumin; CLL, Chronic lymphocytic
leukaemia; CMC, Complement mediated cytotoxicity; CV, Coef-
ficient of variation; FACS, Fluorescence activated cell sorter; FITC,
Fluorescein isothiocyanate; IF, Immunofluorescence; IMDM, Is-
cove’s modified Dulbecco’s medium; MFI, Median fluorescence
intensity; n/a, not applicable; PBS, Phosphate-buffered saline; QC,
Quality control.
*
Corresponding author. Therapeutic Antibody Centre, Old
Road, Headington, Oxford, OX3 7JT, UK. Tel.: +44-1865-744845;
fax: +44-1865-741291.
E-mail address: geoff.hale@path.ox.ac.uk (G. Hale).
www.elsevier.com/locate/jim
Journal of Immunological Methods 260 (2002) 285 – 302