THE RELIABILITY OF THE ANALYTICAL METHODS FOR DETERMINATION OF CONJUGATED ANTIBODIES AFTER FINAL PREPARATION IN SERUM SAMPLES OF RAT

Abstract

Understanding the pharmacokinetics and biodistribution of conjugated antibodies is one of the critical steps in enabling their successful development and use. Their complex structure requires the introduction of multiple bioanalytic methods to define their structure, stability, behavior and fate of both components in vivo.
Appropriate analytical methods should provide insight into the half-life and stability of the blood after application, biodistribution in the body, binding to a specific receptor, time and stability of binding, and the presence of fragments as a result of their destruction.
In our paper we will focus on the method of obtaining conjugated antibodies (rituximab) as well as the use of the HPLC method for their identification in serum samples after intravenous administration. The paper gives us the first indications how the obtained experimental data can be used in the computer simulation of the pharmacokinetic behavior of the same immunoconjugates, to be characterized in biological matrices and to discuss the technical challenges and limitations.
Rituximab (Mabthera®) is an anti-CD20 monoclonal antibody that has demonstrated efficacy in patients with various lymphoid malignancies, including indolent and aggressive forms of B-cell non-Hodgkin s lymphoma (NHL) and B-cell chronic lymphocytic leukaemia (CLL). With the aim of to improve the cytocidal effect of the monoclonals antibodies (mAbs), was introduced the radioimmunotherapy (RIT), where a radioisotope is coupled to a mAb. For labeling mAb with metal and lanthanides radioisotopes, conjugation was previously required in order to introduce a chelating group (p-SCN-Bn-1B4M-DTPA) in the protein chain.
All chemicals and reagents required for experiments were of analytical grade. The antibody solution was previously purified by dialysis or Sephadex gel column. 5-10 mg of antibody was dialyzed against phosphate buffer pH 8.0 and buffer was changed 3 times in 24 hours. The absorbance of a sample of the protein was measured at 280nm in a UV spectrophotometer and protein concentration was calculated in mg/mL.
A rat model was used to monitor the pharmacokinetics of the antibodies and to see their stability after injection. Serum samples were analyzed using the HPLC method and were the first indicator of the possibility that this method would be the first step in their identification.
The structural differences and the behavior of the conjugated antibodies obtained are a major indicator that the use of appropriate analytical techniques, as well as their proper validation and use, is a critical parameter for their development and use as potential therapists.
Only if valid data obtained through appropriate analytical methods can be used as data in computer programs and used for modeling. The HPLC technique is one of the most appropriate, which provides data on the behavior of  conjugated monoclonal antibodies in the blood after their administration and the possibility of using them as a parameter for the role of animal models in translational medicine