The DFO-panitumumab conjugate is used as the authentic standard, which has been prepared following the procedure documented in the literature. The purity was checked using HPLC to compare its retention time with pure authentic panitumumab (Figure 3). The conjugate concentration is determined with the Lowry assay,14 and the DFO to mAb molar ratio is determined with radiometal (89Zr)-binding assay15 where a trace amount of 89Zr is mixed with 25 mmol solution of non-radioactive ZrCl4. This freshly prepared conjugate is then used to develop a standard calibration curve (Figure 4) with HPLC (21 µg/mL to 210 µg/mL of DFO-panitumumab) to determine the DFO-protein concentration in every batch. The amount of mAb per dose and the specific activity of the final product can also be measured with this calibration curve.
Probe Synthesis Protocols
This protocol is for preparing small scale (for one to two patients), clinical-grade 89Zr-panitumumab. The overall process flow of synthesis is shown in Figure 5. Always use sterile and metal-free pipette tips for liquid transfer during the synthesis.
4.1. Bioconjugation and Purification
i) Transfer 0.5 mL (10 mg) of panitumumab from panitumumab vial (100 mg/5mL) to a 1.5 mL microcentrifuge tube (reaction vial).
ii) Adjust the pH of the solution to 9.0 by administering 0.1 M Na2CO3 ( 0.1 mL) with a metal-free pipette tip and pH paper.
iii) Add 0.4 mL 0.9 % saline to make overall volume 1.0 mL
iv) Transfer the ~3 fold molar excess of DFO-Bz-NCS (10 µL of 20 mM DFO stock solution in DMSO) over the molar amount of panitumumab to the reaction vial using a metal-free sterile micropipette tip.
v) Incubate the reaction for 30 to 60 min at 37 °C using a Thermomixer.
vi) Equilibrate a PD10 column with 20 mL of 0.9 % NaCl solution (USP).
vii) Pipette the reaction mixture onto the PD10 column and discard the flow- through.
viii) Pipette 1.5 ml of 0.9% NaCl onto the column and discard the flow-through.
ix) Pipette 2 mL of 0.9% of NaCl onto the PD-10 column and collect the DFO-protein in 0.5 mL fractions. Combine fractions 2 to 4 to get DFO-conjugated protein in a 4 mL glass vial.
4.1.1. In-process QC Tests (Concentration and purity)
i) Transfer 20 µL of the product to a microcetrifuge tube containing 0.5 mL of saline (diluted to 500 times).
ii) Inject 20 µL of this diluted sample to the HPLC instrument.
iii) Integrate the HPLC chromatogram to get the UV peak (280 nm) area.
iv) Calculated protein concentration (mg/ml) using the standard calibration curve (Figure 4).
v) Calculate the volume needed for 1 mg protein.
4.2. Radiolabeling with 89Zr
i) Pipette the required volume (4-5 mCi, ~ 250 μL) of 89Zr oxalic acid solution into a reaction vial.
ii) Calibrate the reaction vial using a dose calibrator to determine the amount of activity in mCi.
iii) Pipette an adequate amount of 2M Na2CO3 (~100 μL) into the reaction vial to incubate for 3 min at room temperature. Adjust pH of reaction solution to 7.5 using 2M Na2CO3.
iv) While gently shaking add: a) 0.5 ml 0.5M HEPES (pH7.2); b) adequate volume of conjugate to get 1-1.2 mg mass of the conjugate; c) 0.2 mL gentisic acid solution.
v) Incubate the reaction for 1 h at room temperature with occasional gentle shaking.
4.2.1. Radiochemical Yield (RCY) of Crude Product
i) Dilute approximately 2 μL of crude product to 500 times with 0.9% NaCl.
ii) Perform the ITLC experiment by pipetting ~ 1µL of diluted sample to the ITLC plate.
iii) Develop the ITLC plate in the developing chamber using 20 mM of citric acid solution as the mobile phase.
iv) Cut the plate in middle and count both the bottom and top parts separately for radioactivity in a gamma well counter.
v) Calculate RCY using this equation
RCY\(%) = \(counts of the bottom / counts of \(top + bottom)) X 100
vi) If RCY is < 50%, discard the whole batch and start new labeling reaction.
4.2.2. Purification of 89Zr-Panitumumab
i) Rinse/equilibrate a PD-10 column with 20 mL of the mobile phase.
ii) Pipette the conjugation reaction mixture onto the column and discard the flow-through.
iii) Pipette 1.5 mL of the mobile phase onto the column and discard the flow-through.
iv) Pipette 2 mL of the mobile phase onto the PD-10 column and collect the 89Zr-DFO-protein at 0.5 mL and 1.5 mL fractions in small glass vials. Check amount of activity of each fraction in dose calibrator.
v) Pipette 0.5 mL of the mobile phase onto the PD-10 column and collect it as 3rd fraction. Check amount of activity in dose calibrator.
vi) Check the radiochemical purity of fraction two and three with ITLC.
vii) Combine fractions two and three in a 10 mL vial if they are > 90% pure. Check the amount of radioactivity using a dose calibrator.
4.2.3. Sterile Filtration of the Final Product
i) Dilute the product to 5 mL using 0.9% saline.
ii) Draw the product solution in a 10 mL syringe.
iii) Remove the needle and attach a sterile filter.
iv) Connect the filter needle to a 10 mL sterile vial with a venting needle.
v) Pass the solution through the sterile syringe filter slowly by pressing the plunger of the syringe.
vi) Perform bubble point test of the sterile filter following the standard procedure.
vii) Detach the product vial from the syringe attached to the sterile filter.
4.3. Post-Synthesis QC Test Protocols.
All the QC tests, except the sterility test of 89Zr-panitumumab, were carried out according to the USP recommendations as detailed below. After successfully meeting all release criteria, doses are released to physicians for human administration. A post-release sterility test for every batch must be completed and recorded.16
4.3.1. Sampling for Quality Assurance.
i) Assay the product vial for total radioactivity in a dose calibrator.
ii) Visually inspect for particulates and color. The final drug product in the vial should be clear and colorless, without any visible particulates as per USP chapters 823 and 631 on color and achromaticity.
iii) Complete the integrity test of the sterilizing filter.
a) Place the sterilizing filter on a gas line with a pressure gauge and the outlet of the filter under water.
b) Increase the gas pressure slowly on the inlet to the filter until a steady stream of bubbles is observed at the filter outlet.
c) The pressure at which the bubble stream begins is recorded and compared with the manufacturer’s pressure rating ( ≥ 46 psi) for the filter (Pall Corp. Ann Arbor, MI).
iv) After a successful filter integrity test withdraw 300 µL of the product for further QC and sterility testing.
4.3.2. Chemical and Radiochemical Purity/Identity by HPLC.
The purity of the drug product can be determined by HPLC analysis.
i) A blank injection must be completed before the sample is injected to make sure there are not any UV and/or radiation impurities from the column or the instrument.
ii) Inject 20 µL of 89Zr-panitumumab (auto-injection preferable).
iii) Integrate all of the UV and radiation responses to obtain the peak areas for the [89Zr]-panitumumab.
iv) Perform the reverse correlation from the linear equation of DFO-panitumumab to determine the concentration of [89Zr]-panitumumab in the final solution.
Typically, the final concentration of [89Zr]-panitumumab in the [89Zr]-panitumumab product to be tested will be < 0.2 mg/mL.
v) Any other 280 nm UV absorbing chromatographic peaks that elute after 3.0 minutes and before 40 minutes post injection should be integrated individually and shown as a sum of less than “10%” in the final product. Assume that the molar UV absorption coefficients for the unknown contaminants are the same as those for [89Zr]-panitumumab, and that the [89Zr]-panitumumab standard curve is applicable to the unknown contaminants.
vi) Integrate all of the radioactive peaks in the chromatogram and calculate the percent of the [89Zr]-panitumumab radioactivity in the final product. The radioactivity of [89Zr]-panitumumab in the final product should be more than 90%.
4.3.3. Radiochemical Purity by ITLC.
i) Transfer 0.5 mL of the ITLC eluent to a 15 mL centrifuge tube (developing chamber).
ii) Using a micro pipette spot ~1 µL of sample at the origin of the ITLC strip.
iii) Allow the spot to dry prior to placing it in developing chamber.
iv) Develop the ITLC plate by carefully positioning it in the developing chamber.
v) Allow the solvent front to migrate at least 90% of the length of the ITLC plate.
vi) Cut the ITLC plate into the middle and place each part in separate counting tubes.
vii) Count for radioactivity with an appropriate counter (Perkin-Elmer gamma counter).
viii) Calculate the radiochemical purity (RCP) using this equation
RCP\(%) = \(counts of the bottom / counts of \(top + bottom)) X 100.
ix) The acceptable RCP should be ≥ 90%.
4.3.4. pH Determination.
Because the product volume is small and the product is radioactive, pH test strips are used instead of a pH meter.
i) The pH test strips are checked by pipetting pH 5 and pH 7 calibrated commercial pH standards onto individual strips.
ii) The color on the strips must match the pH 5 and pH 7 on the color key supplied with the test strips.
iii) Then the 89Zr-panitumumab is pipetted onto another test strip, and the color is checked against the color key.
iv) The measured pH must be between pH 6 to 8.
4.3.5. Bacterial Endotoxin Test.
The levels of bacterial endotoxin in 89Zr-panitumumab were tested and qualified by PTS Limulus Amebocyte Lysate (LAL) Test using Endosafe®-PTS (portable test system) from Charles River Laboratories, as described in USP. All of the bacterial endotoxin levels were < 175 EU per batch for the initial qualification syntheses. This testing required approximately 15 min.
4.3.6. Radionuclidic Identity
Half-life determination is utilized for verifying the radionuclidic identity of the final product. The USP radioactivity general chapter, 821, states that the half-life can be “readily determined by successive counting of a given source of a radionuclide over a period of time that is long compared to its half-life.” The variation used here is to count for only a fraction of the half-life of 78 hours. To count a sample for more than 78 hours would reduce the radiopharmaceutical dose by more than half.
For the test, an aliquot of the product 89Zr-panitumumab or 89Zr-oxalate (used for this synthesis) is counted in an ion chamber or gamma counter at least three times at a particular time-point. The half-life of the radioactivity is determined for each activity measurement using the following equation.
The half-life test result for 89Zr must be between 74 and 82 hours for the dose to pass.
4.3.7. Sterility Test
Sterility testing can be performed using the direct inoculation method, which is required by USP after releasing the product for human administration. This test requires ~14 days of time.