Conjugation of chelating agents to proteins and radiolabeling with trivalent metallic isotopes

Abstract

Peptides and proteins may be tagged with metallic elements in order to use them as imaging reporters or for other applications. The polypeptide of interest is first conjugated to a suitable chelating agent that forms stable complexes with the element of interest. This conjugation step is undertaken either in aqueous or in non-aqueous conditions depending on the solubility of the substrate. For polypeptides of greater than approximately 10 kDa in size, this is normally done in aqueous medium. Most commonly the chelators are reacted with lysine amino groups. The protein is first desalted into a suitable buffer at pH 8-9 and a molar excess of a bifunctional chelating agent is added. After a suitable period of incubation, excess, unreacted or hydrolyzed chelator is removed and the protein conjugate is desalted into an acidic buffer. The conjugate can then be tagged by addition of a suitable metal salt followed, if necessary, by removal of unchelated metal. As described in the protocol that follows, the entire conjugation, purification and labeling procedure takes about 2 d.

Comparison of 64Cu-Complexing Bifunctional Chelators for Radioimmunoconjugation: Labeling Efficiency, Specific Activity, and in Vitro/in Vivo Stability

“…64 Cu (t 1/2 = 12.7h), with both β + and β − emissions, allows for both PET imaging and radionuclide therapy, and 64 Cu labeled antibodies have attracted considerable interest in the field of targeted radionuclide therapy and diagnosis. In addition, copper has several other medically relevant isotopes ( 60 Cu, 61 Cu, 62 Cu and 67 Cu) that are potentially useful for either diagnosis or therapy 2,3 . Although direct labeling approaches for labeling antibodies with 64 Cu have been proposed 4 , indirect labeling using a BFC is the preferred method to control stability of the 64 Cu-antibody complex formed.…”

Development of radioimmunoconjugate for diagnosis and management of head-and-neck subclinical cancer and colorectal carcinoma

“…Considering an initial antibody concentration of 5 mg/mL, it obtained a protein recovery above 83±1.42% (N = 15). These results demonstrated that the method applied for purification of immunoconjugate was more reproducible than the method described in the literature (Cooper, Sabbah, Mather, 2006), which results are variable due to the loss of protein (10 -50%).…”

“…The distribution of radioactivity in the chromatographic plate was performed by radiochromatography (radio-TLC Imaging Scanner AR-2000, Eckert & Ziegler / USA) (Chang, Smith, Lapi, 2013). The radiochemical purity was also evaluated by HPLC (Agilent, detector UV 190-300 nm) (Cooper, Sabbah, Mather, 2006;Chang, Smith, Lapi, 2013), using the same methodology as described above.…”

“…The production of radiolabeled monoclonal antibodies is a process that involves several steps (Cooper, Sabbah, Mather, 2006): (1) pre-purification of mAb to remove free metals; (2) conjugation of mAb with chelating groups; (3) immunoconjugate purification and concentration; (4) and radiolabeling.…”

Positron Emission Tomography/Computed Tomographic and Magnetic Resonance Imaging in a Murine Model of Progressive Atherosclerosis Using 64 Cu-Labeled Glycoprotein VI-Fc

“…11 Briefly, using this method, the protein is stripped of metal ions using EDTA and then the buffer exchanged into 0.1 mol/L HEPES buffer pH 8.9 by ultracentrifugation. p-SCN-Bn-NOTA (as a 40-fold molar excess to protein) in dimethyl sulfoxide is added, and the conjugation is allowed to proceed at ambient temperature for 3 hours.…”