A kit to prepare In-111-DTPA-trastuzumab (Herceptin) Fab fragments injection under GMP conditions for imaging or radioimmunoguided surgery of HER2-positive breast cancer

作者：Scollard Deborah A; Chan Conrad; Holloway Claire M B; Reilly Raymond M^*

来源：Nuclear Medicine and Biology, 2011, 38(1): 129-136.

DOI：10.1016/j.nucmedbio.2010.06.010

• Breast cancer
• Trastuzumab Fab
• In-111
• kit
• Good manufacturing practices (GMP)
• Radioimmunoguided surgery (RIGS)
• Imaging

Introduction: The human epidermal growth factor receptor-2 (HER2) gene is amplified in 25% of invasive breast cancers, and receptor overexpression has been noted in up to 60% of early stages of the disease [ductal carcinoma in situ (DCIS)]. Preclinical studies have revealed high tumor/blood ratios (>27:1) for In-111-labeled Fab fragments of the HER2 monoclonal antibody, trastuzumab (Herceptin) (In-111-DTPA-trastuzumab Fab) at 72 h pi in athymic mice bearing subcutaneous human breast cancer xenografts. Our aim in this study was to formulate a kit for preparation of In-111-DTPA-trastuzumab Fab injection under good manufacturing practice (GMP) conditions suitable for human administration in a Phase I clinical trial of imaging and radioimmunoguided surgery (RIGS) of HER2-positive breast cancer.
Methods: Fab fragments were produced by digestion of trastuzumab IgG (Herceptin) with immobilized papain for 20 h at 37 degrees C. Fab fragments were purified by ultrafiltration, then reacted with a 10-fold molar excess of diethylenetriaminepentaacetic acid (DTPA) dianhydride. DTPA-Fab fragments were purified,, then sterilized by filtration into unit dose glass vials (kits). Kits were tested against specifications for volume (0.9-1.1 ml), protein concentration (0.45-0.55 mg/ml), pH (5.5-6.5), DTPA substitution (0.5-4.0 mol DTPA/mol Fab), appearance (clear, colorless and particle free), labeling efficiency (>= 85%), and sterility and apyrogenicity (USP XXXII). Immunoreactivity of In-111-DTPA-trastuzumab Fab towards HER2 was measured by saturation radioligand binding assays using SKBR-3 human breast cancer cells (specifications: K-a=0.6-9.6x10(7) L/mol; B-max=0.6-10.4x10(6) sites/cell). In-111-DTPA-trastuzumab Fab injection was prepared by adding 80-100 MBq of (InCl3)-In-111 to a single kit vial and incubating for 30 min at room temperature. In-111-DTPA-trastuzumab Fab was assayed for the amount of radioactivity and tested for pH, radiochemical purity (RCP), appearance and sterility.
Results: Pure and homogeneous Fab fragments were produced. Eleven lots of kits met established quality specifications. The labeling efficiency with In-111 was 90.6 +/- 2.2%. In-111-DTPA-trastuzumab Fab bound specifically to HER2 on SKBR-3 cells (K-a=4.8 +/- 2.5x10(7) L/mol and B-max=1.6 +/- 0.8x10(6) sites/cell). Thirteen lots of In-111-DTPA-trastuzumab injection met all established specifications. Kits were stable for 90 days and In-111-DTPA-trastuzumab Fab injection was stable for 24 h stored at 4 degrees C.
Conclusions: A kit was formulated under GMP conditions for the preparation of In-111-DTPA-trastuzumab Fab injection suitable for human administration. The kits were approved by Health Canada.

• 出版日期2011-1