Gel electrophoresis-autoradiographic image analysis of radiolabeled protein drug concentration in serum for pharmacokinetic studies.
BACKGROUND
The purpose of this study was to evaluate the feasibility of using gel electrophoresis combined with autoradiographic image analysis for quantitating protein drug concentrations in biological fluid for pharmacokinetic studies.


METHODS
Protein drugs were iodinated using the Iodogen reagent and injected into Sprague-Dawley rats for pharmacokinetic evaluation. Serum samples were analyzed using trichloroacetic acid (TCA)-precipitable counts or sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Commercially available precasted Bis-Tris gradient gels were used for SDS-PAGE. Autoradiography of gel samples was performed using phosphoimager and quantitated using the ImageQuant software.


RESULTS
The maximum loading volume for protein drugs with molecular weight close to that of albumin ( approximately 70 kDa) was about 1 microl, whereas for protein drugs with larger or smaller molecular weight (i.e., >80 or <40 kDa), the maximum loading volume was up to 20 microl/lane. The optimal exposure time was about 18 h or overnight. Standard curves were constructed using serially diluted dosing solution, which was linear over a 10-fold concentration range with a correlation coefficient of.98. Comparing to the soluble human interleukin-13 receptor (shIL-13R) pharmacokinetic profiles from TCA-precipitable counts, the quantitative gel analysis revealed lower concentrations at later time points and a lower bioavailability from intraperitoneal injection.


CONCLUSIONS
This study provided the first systemic evaluation of gel electrophoresis technology for quantitative protein drug determination in serum and its application in pharmacokinetic studies. The combination of gel electrophoresis with autoradiographic image analysis provided accurate and specific quantitation results. The overnight turnover time allowed routine application in preclinical pharmacokinetic studies.
Song
Di
D
Drug Safety and Metabolism, Wyeth Ayerst Research, Andover, MA 01810, USA. dsong@wyeth.com
Ma
Shang
S
Khor
Soo Peang
SP
D000818:Animals; D001345:Autoradiography; D001709:Biotechnology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007275:Injections, Intravenous; D053661:Interleukin-13 Receptor alpha1 Subunit; D007457:Iodine Radioisotopes / Q000097:blood; D051381:Rats; D017207:Rats, Sprague-Dawley; D018123:Receptors, Interleukin; D053660:Receptors, Interleukin-13; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000097:blood / Q000493:pharmacokinetics*
C508181:IL13RA1 protein, human; C508185:Il13ra1 protein, rat; D053661:Interleukin-13 Receptor alpha1 Subunit; D007457:Iodine Radioisotopes; D018123:Receptors, Interleukin; D053660:Receptors, Interleukin-13; D011994:Recombinant Proteins