α-Labeling of J591, an Antibody Targeting Prostate-Specific Membrane Antigen: The Technique and Considerations from the First Dedicated Production Lab at an Academic Institution in the United States

TECHNICAL AND PRODUCTION DETAILS

Production Assembly Steps

A 37 MBq (1.0 mCi) activity of ²²⁵Ac-nitrate residue is supplied in a 2-mL glass vial (Department of Energy, Oak Ridge National Laboratory). Figure 1 summarizes the production assembly mechanism.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 1.

Steps for production assembly of ²²⁵Ac-J591. DG = durable grade; DTPA = diethylenetriaminepentaacetic acid; FDP = final drug product; HSA = human serum albumin; SDP = sterile drug product; SWFI = sterile water for injection; TMAA = triethylammonium acetate.

A 30-mL 10 durable grade (10 DG) desalting disposable column is washed twice with 15 mL of 3% hydrogen peroxide in a laminar flow hood. Thirty minutes later, the column is washed twice with 15 mL of sterile water for injection and twice with sterile 0.9% saline with 2% human serum albumin. This column is prepared for purification of the radiolabeled mAb. Within the same laminar flow hood, a 20-gauge sterile needle is attached to a 0.2-μm sterile filter and inserted into a pyrogen-free sterile drug product vial and the final drug product vial.

Using aseptic technique in a laminar flow hood, 100 μL of 0.2 M hydrogen chloride are added to the ²²⁵Ac vial for 30 min; 3,700–5,550 kBq (100–550 μCi) are removed and added to the reaction vial; and 75–125 μL of 2 M triethylammonium acetate buffer, 25 ± 5 μL of ascorbic acid, and 3 mg of DOTA-HuJ591 mAb (300 ± 15 μL) are subsequently added to the vial. The DOTA labeling of the HuJ591 mAb is performed at an outside facility. After gentle mixing of the vial, it is incubated at 37°C for 1–2 h. Instant thin-layer chromatography with silica gel is performed to determine labeling efficiency, which must be more than 50% to be acceptable for further processing.

Diethylenetriaminepentaacetic acid solution, 50 μL, is added to the vial and incubated for an additional 10 min at 37°C. The reaction mixture is loaded onto the prepared desalting column, and the eluate is collected into a sterile vial (fraction 1). The reaction vial is rinsed with 1.5 mL of 2% human serum albumin in sterile saline and reloaded in the column, and the eluate in the fraction 1 vial is collected. Fraction 2 is eluted with 4 mL of sterile saline with 2% human serum albumin into a sterile tube. Using a sterile syringe, 4 mL of fraction 2 are removed and transferred through a sterile filter into the final drug product vial. The filter is rinsed with 1.0 mL of saline–human serum albumin solution. The activity of ²²⁵Ac-DOTA-HuJ591 mAb over the next 6–8 h is measured at intervals of 1–2 h.

The ²²⁵Ac-DOTA-J591 mAb injection is stored in a 10-mL sterilized vial sealed with gray butyl rubber septa crimped closed with an aluminum stopper (Hollister Stier Laboratories or Allergy Laboratories) and certified as sterile and apyrogenic.

QUALITY CONTROL AND QUALITY ASSURANCE TESTING

Each batch of the drug product produces 1–2 doses of ²²⁵Ac-DOTA-HuJ591 mAb injection. Before administration, several quality control and quality assurance tests are performed and assembled in a report to ensure that each batch meets the institutional acceptance criteria for batch release (Table 1).

The final drug product must be colorless after being swirled in the glass vial, suggesting no contamination. Instant thin-layer chromatography with silica gel and 10 mM ethylenediaminetetraacetic acid solution determines the radiochemical purity of the drug batch, which must be more than 95% (Fig. 2). The labeling efficiency before ethylenediaminetetraacetic acid elution is remarkable for a high concentration of ²²⁵Ac bound to protein and a small percentage of unbound ²²⁵Ac. Unbound ²²⁵Ac will decay to ²¹⁸Fr and ⁴⁴⁰Bi daughter compounds, the greatest concentration of which appear at 2 h after elusion. Because of the shorter half-lives of the daughter compounds, their levels are negligible at 24 h after elusion. Figure 3 provides a schematic explanation of ²²⁵Ac decay.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 2.

Labeling efficiency and radiochemical purity measured using instant thin-layer chromatography. (A) Labeling efficiency before ethylenediaminetetraacetic acid elusion, remarkable for high concentration of ²²⁵Ac bound to protein (green peak) and small percentage of unbound ²²⁵Ac, which has formed into daughter compounds of francium and bismuth (red peaks). (B and C) Radiochemical purity after ethylenediaminetetraacetic acid elusion at 2 h (B) and 24 h (C). Two hours after elusion, higher concentration of daughter compounds is noted whereas peak concentration of ²²⁵Ac bound to protein is stable. Because of shorter half-lives of daughter compounds, their presence is negligible at 24 h after elution, validating chemical purity of final product.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 3.

Schematic diagram of ²²⁵Ac decay and daughter compounds.

The radionuclide concentration is measured with a dose calibrator. The pH is measured by placing 2–5 μL of aliquot on pH indicator paper. The specific activity is estimated through dose calibrator measurement of total activity relative to the 3 mg of the precursor DOTA-HuJ591 used. Endotoxin content is measured using the Endosafe-PTS system (Charles River) according to manufacturer’s operation manual, with a goal of not exceeding 175 endotoxin units for the entire 5-mL batch of the final drug product (35 endotoxin units/mL) (Fig. 4).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 4.

Endosafe-PTS test record. Endotoxin content should demonstrate limit well within guidelines of 175 endotoxin units for entire batch, as demonstrated here. Cal = calibration; CV = coefficient of variation; Rxn = reaction; Sec = seconds.

The bubble point test in a nondestructive test evaluating filter integrity. It determines the minimum pressure required to force liquid out of the filter pores, which is an indirect way of measuring the pore diameter and, in essence, any defects in the filter that may permit bacterial penetration. The pressure at which bubbles appear must be greater than the minimum acceptable bubble point pressure as defined by the manufacturer. Fluid thioglycolate medium and soybean casein digest medium were used to evaluate the sterility of the injection drug product by transferring 0.1-mL samples of ²²⁵Ac-DOTA-J591 to the medium and incubating either at 37°C (fluid thioglycolate medium) or at room temperature (soybean casein digest medium) up to 14 d. Growth in the medium through visual inspection is recorded on the third, seventh, and 14th days after culturing.

²²⁵Ac-J591 can be stored at 4°C–8°C for up to 4 h after production, which also represents the time window for therapeutic use of the produced dose.

RADIATION SAFETY

Lead-shielded waste containers and sharps disposal containers should be close to where the ²²⁵Ac work is performed. A spill kit must be present in the room. An absorbent pad must be placed under the production and distribution area for ²²⁵Ac, including the laminar hood and glove box. Gloves, booties, and gowns must be worn by staff during production. Gloves should be changed frequently and placed in a leaded waste container.

If the staff needs to leave the room, their feet and hands must be checked using a survey meter (e.g., Ludlum model 14C). Similarly, when tagging is complete, the hands, feet, and body of each individual need to be evaluated for radiation contamination. If there is evidence of contamination, the survey meter should be used to check the area. Also, equipment used for tagging must be checked for contamination after each use. If contamination is found, the area must be decontaminated and checked to ensure that radiation is at background levels. Additionally, a wipe test must be done weekly using a γ-counter following a survey map.

THERAPEUTIC ADMINISTRATION CONSIDERATIONS

The equipment needed is a 50-mL 0.9% sterile sodium chloride infusion bag, sterile Alaris tubing intravenous sets (with a clamp to regulate or stop flow), two 3-way stopcocks, a spinal needle, an 18-gauge needle, a peristaltic infusion Alaris pump, and clamps. The spinal needle and 18-gauge needle are inserted into the therapeutic vial. The spinal needle is connected to intravenous tubing within the infusion pump. The output of the intravenous tubing is connected to a 3-way stopcock and then to the patient’s intravenous line. The 18-gauge needle is connected to the 50-mL 0.9% sterile sodium chloride infusion bag via a 3-way stopcock (which remains clamped until the end of the infusion).

The pump is set to 250 mL/h at the beginning of the administration. Within 10 min, the therapy vial solution is nearly empty. A saline rinse is begun by unclamping the 50-mL 0.9% sterile sodium chloride infusion bag. The rinse helps remove the solution from the vial and all lines to optimize dose delivery by minimizing residual agents in the equipment.

CLINICAL CONSIDERATIONS

Current guidelines require that individuals with metastatic castration-resistant prostate cancer who have refractory disease or are unwilling to undergo other commercially available prostate cancer therapies should be considered for experimental treatment with ²²⁵Ac-J591. ²²⁵Ac-J591 is metabolized by the liver, as well as excreted by the kidney and the bowel. Therefore, baseline serologies evaluating hepatic and renal function must be reviewed before administration of the radioimmunotherapeutic agent. Not much uptake is seen in the marrow by default, but it is imperative to also review blood counts. PSMA PET/CT imaging may also be used to evaluate the baseline tumor burden before administration of the ²²⁵Ac-J591 (45 kBq/kg). After the treatment course, currently performed at our institution as 2 sessions 6 wk apart, repeat imaging helps guide clinicians in planning further management (Fig. 5).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 5.

PSMA PET imaging demonstrating good response in this patients with metastatic castration-resistant prostate cancer at baseline (B) and after receiving 2 sessions of ²²⁵Ac-J591 (A). Marked decrease in metastatic disease burden is demonstrated in maximum-intensity-projection images, particularly in subcarinal lymph node (column 2) and osseous lesions (column 3).

DISCLOSURE

No potential conflict of interest relevant to this article was reported.