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Randon M. Burr, Sarah A. Molokhia, Jacquelyn M. Simonis, Nathan Gooch, Barbara M. Wirostko, Balamurali K. Ambati; Conjugation of Gadolinium Based Contrast Agent to Avastin for Pharmacokinetics with MRI. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3250.
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To conjugate Gd-DTPA to the drug Avastin®.
The use of zero length cross linkers was employed to derivatize Gd-DTPA (Sigma) to Avastin® (Genentech). Gd-DTPA was dissolved in MES of pH 6.0 at 1 mg/ml. EDC and S-NHS (Thermo Scientific) were added to the solution at concentrations of 2 mM and 5 mM respectively. The solution was gently spun for 30 minutes at room temperature. The pH was raised to approximately 7.0 - 7.4 by the addition of 5M NaOH. Immediately following, Avastin® is added to a concentration of 1 mg/ml and the solution is mixed for 24 hours at room temperature. The solution was diluted to 15 ml and then centrifuged at 4000 g for 20 minutes in a 30 kDa MWCO centrifugal filtrate unit (Millipore) to remove any unconjugated Gd-DTPA and unreacted maleamides. The same centrifugation process was repeated once more. SEC - HPLC was used to confirm the addition of the Gd-DTPA with Superdex 200 10/30 GL column (GE Healthcare) and a mobile phase of PBS pH 7.0 at 0.5 mL/minute. ICP-OES was used to quantify the Gd content in solution and calculate a theoretical binding ratio of Gd-DTPA molecules per Avastin® molecule. Alterations to the binding affinity were identified using ELISA and integrity of the Avastin® was analyzed using IEX-HPLC with a bio MAb column (Agilent).
We were able to detect peak shifts in retention time using SEC of approximately 1 - 2 minutes; the change in time depends upon the reaction conditions. After the use of the spin filter units the presence of Gd was detected. Our group has previously reported the development of a non-degradable drug delivery device for implantation in the capsular bag at the time of cataract surgery. We intend to develop a formulation with the derivatized Avastin® suitable for release from our drug delivery device.
The results suggest the addition of the Gd-DTPA to the Avastin® molecules. We intend to use this formulation to conduct pharmacokinetics analysis of Avastin® using MRI. Further characterization with mass spectrometry can confirm the exact number of Gd-DTPA molecules added per Avastin® molecule. Additional characterization using the combination of IEX and ELISA will be used to confirm the dervatized drug’s stability.
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